Temperature correlated force and structure development of elastin polytetrapeptides and polypentapeptides

ABSTRACT

A bioelastomer containing elastomeric units comprising tetrapeptide, or pentapeptide or units thereof modified by hexapeptide repeating units and mixtures thereof, wherein said repeating units comprise amino acid residues selected from the group consisting of hydrophobic amino acid and glycine residues, wherein said repeating units exist in a conformation having a β-turn which comprises a polypentapeptide unit of the formula: 
     
         --X.sup.1 --(IPGVG).sub.n --Y.sup.1 -- 
    
     wherein 
     I is a peptide-forming residue of L-isoleucine; 
     P is a peptide-forming residue of L-proline; 
     G is a peptide-forming residue of glycine; 
     V is a peptide-forming residue of L-valine; and 
     wherein X 1  is PGVG, GVG, VG, G or a covalent bond; Y 1  is IPGV, IPG, IP, I or a covalent bond; and n is an integer from 1 to 200, or n is 0, with the proviso that X 1  and Y 1  together constitute at least one of said pentameric unit, in an amount sufficient to adjust the development of elastomeric force of the bioelastomer to a predetermined temperature.

BACKGROUND OF THE INVENTION

The government has rights in this invention as a result of the workdescribed herein being supported in part by the National Institutes ofHealth under Grant No. HL-29578.

FIELD OF THE INVENTION

This invention relates to bioelastomers, particularly to bioelastomerswhich can be used as replacements for elastin, and more particularly tobioelastomers which exhibit controllable elastomeric force developmentas a function of temperature.

DESCRIPTION OF THE BACKGROUND

The connective tissue of vascular walls is formed from two principaltypes of protein. Collagen, in general, the principal proteinaceouscomponent of connective tissue, constitutes the structural elementimparting strength to the tissue. However, where the demand forelasticity is great as in the aortic arch and descending thoracic aorta,there is twice as much elastin as collagen. In the vascular wall, andparticularly in the internal elastic lamina thereof, collagen isassociated with natural elastic fibers formed from a different type ofprotein, known as tropoelastin. In the relaxed vascular wall, collagenfibers tend to be folded or crimped, and the elastic fibers are in aretracted state. Upon distention or stretching, the elastic fibersbecome stretched, and, before their extension limit is approached, thecollagen fibers come into tension to bear the load. As the loaddiminishes, the elastic fibers draw the wall back to its originaldimension and the collagen fibers back into a folded state.

The above can also be demonstrated experimentally, for if the collagencomponent of an intact ligament is removed in vitro by the enzymecollagenase, the resultant stress-strain relationship clearly indicatesthat the elastic component, elastin, is principally responsible for theinitial high yield response of the intact ligament. Conversely, removalof elastin by the enzyme elastase leaves collagen which is observed tobe responsible for only the final portion of the response of the intactligament. See Introductory Biophysics, F. R. Hallett et al. (HalstedPress, 1977).

Presently available synthetic vascular materials, such as Dacron, arequite different from natural connective tissue in that the syntheticweave can be viewed as providing the structural analog of foldedcollagen, but there is no true elastomeric component therein.

The central portion of the elastic fibers of vascular wall, skin, lungand ligament is derived from a single protein called tropoelastin.Elastin, the actual elastomeric component of biological elastic fibers,is composed of a single protein and is formed from the cross-linking ofthe lysine residues of tropoelastin. The sequence of elastin can bedescribed as a serial alignment of alanine-rich, lysine-containingcross-linking sequences alternating with glycine-rich hydrophobicsequences. More than 80% of the elastin sequence is known, and it hasbeen shown that vascular wall tropoelastin contains a repeat hexapeptide(Ala-Pro-Gly-Val-Gly-Val)_(n), a repeat pentapeptide(Val-Pro-Gly-Val-Gly)_(n), and a repeat tetrapeptide(Val-Pro-Gly-Gly)_(n) where Ala, Pro, Val and Gly, respectively,represent alanine, proline, valine, and glycine amino acid residues.These residues can also be represented, respectively, as A, P, and G,inasmuch as amino acids can be referred to either by standardthree-letter or one-letter abbreviations. See, for example, OrganicChemistry of Biological Compounds, pages 56-58 (Prentice-Hall, 1971).Further, in this application, all peptide representations conform to thestandard practice of writing the NH₂ -terminal amino acid residue on theleft of the formula and the CO₂ H-terminal amino acid residue on theright. Furthermore, unless otherwise specified all amino acids are ofthe L-configuration, with the exception of Glycine, which is opticallyinactive.

The nature of the amino acid sequence in the vicinity of thetropoelastin cross-links is also known. Moreover, a high polymer of thehexapeptide has been synthesized, and found to form cellophane-likesheets. In view of this, and its irreversible association on raising thetemperature in water, the hexapeptide is, therefore, thought to providea structural role in the natural material. On the other hand, synthetichigh polymers of the pentapeptide and of the tetrapeptide have beenfound to be elastomeric when cross-linked and have the potential tocontribute to the functional role of the elastic fiber. In fact, thechemically cross-linked polypentapeptide can, depending upon its watercontent and degree of crosslinking, exhibit the same elastic modulus asnative aortic elastin.

More recently, a synthetic polypentapepide based on the pentapeptidesequence disclosed above was disclosed and claimed in U.S. Pat. No.4,187,852 to Urry and Okamoto. Furthermore, a composite bioelasticmaterial based on an elastic polypentapeptide or polytetrapeptide and astrength-giving fiber was disclosed and claimed in U.S. Pat. No.4,474,851 to Urry. Additionally, a bioelastic material having anincreased modulus of elasticity formed by replacing the third amino acidin a polypentapeptide with an amino acid of opposite chirality wasdisclosed and claimed in U.S. Pat. No. 4,500,700 to Urry and to anenzymatically cross-linked polypeptide as disclosed in and claimed inU.S. Pat. No. 4,589,882. Finally, it is also noted that at present, Ser.No. 533,670, directed to a chemotactic peptide and Ser. No. 793,225,directed to a second chemotactic peptide are both pending. Also pendingis Ser. No. 853,212, directed to a segmented polypeptide bioelastomerfor the modulation of elastic modulus.

At present, there is a tremendous demand for new synthetic vascularmaterials and prostheses. Hence, there is a consequent demand for newbioelastic materials based on the above-described polypentapeptide andpolytetrapeptide repeating sequences which have desirable, but modifiedchemical and biological characteristics. This demand is, perhaps, due tothe ubiquitous nature of elastin in the human body and the implicationsthereof. For example, in the extracellular matrix of the vascular wall,the elastin fiber is a primary site of lipid deposition contributing tothe gruel of atherosclerosis. Further, in pulmonary emphysema, elastinfibers are disrupted and rendered dysfunctional. Additionally, it can benoted that there are many disease states involving elastin fibers anddysfunctions thereof, for example, the heritable disorders such aspseudoxanthoma elasticum, cutis laxa, endocardial fibroelastosis, andthe Buscke-Ollendorf, Ehlers-Danlos, Menkes, and Marfans syndromes.Furthermore, elastin fiber dysfunction is also implicated in theacquired diseases: actinic elastosis, isolated elastomas, elastofibromadorsi and elastosis perforans serpiginosa. Even from a purely cosmeticstandpoint, it is known that solar elastosis of the dermis contributesto the wrinkles of age, and underlying the wrinkles, the elastin fibersare found to be ruptured. Clearly, the development of new syntheticpolypentapeptide and polytetrapeptide elastomers would provide, for thefirst time, versatile substitutes for damaged natural elastin fibers aswell as new methods for treating these various diseases.

However, until recently, little has been known about the elasticproperties of the bioelastomeric polytetrapeptides andpolypentapeptides. Thus, the rational design of specific bioelastomersfor particular structural purposes has been extremely limited. Forexample, up until the present, it has not been possible to vary thetemperature range over which would occur the elastomeric forcedevelopment of synthetic bioelastomers. It would seem that such controlwould be imperative in order to rationally design a suitablebioelastomeric material for a given purpose. For example, in order todesign a thermomechanical transducer for a predetermined temperature, itis necessary to provide materials which development elastomeric forcewithin different temperature ranges.

It is difficult to underestimate the importance of selecting the rightmaterial for a particular biological or industrial function. Forexample, in Technology Review, Nov./Dec. 1984 (Edited at MIT), it wasnoted that a major obstacle to the development of a reliable artificialheart, as well as prosthetic devices generally, was the lack of suitablesynthetic biomaterials. In the case of the artificial heart, it wasfound that calcium was deposited therein to an unacceptable extent,among other problems. Quite appropriately, Bronowski has noted in hisOlympian work the Ascent of Man, that:

In effect the modern problem is no longer to design a structure from thematerials but to design materials for a structure.

However, before bioelastic materials can be rationally designed forparticular biological purposes requiring variable elasticity, it will benecessary to provide a means for rationally controlling the elastomericforce development of the bioelastomer. Thus, it would be extremelydesirable to provide a means for controlling the elastomeric forcedevelopment of the bioelastomers as a function of temperature. Thiswould greatly broaden the variety of environments in which suchmaterials could function. At present, no such control is possible.

Accordingly, in general, a need clearly continues to exist forbioelastic materials based on polypentapeptide and polytetrapeptiderepeating sequences which exhibit desirable chemical and biologicalcharacteristics. In particular, a need continues to exist for suchbioelastic materials, the elastomeric force development of which can becontrolled and varied as a function of temperature.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provideelastomeric polymers which exhibit an elastomeric force developmentwhich can be varied as a function of temperature.

Additionally, it is also an object of this invention to provide a methodof making elastomeric polymers which exhibit elastomeric forcedevelopment which can be varied as a function of temperature.

These and other objects of the invention as will hereinafter become morereadily apparent have been accomplished, in part, by providing abioelastomer containing elastomeric units comprising tetrapeptide,pentapeptide and units thereof modified by hexapeptide repeating unitsand mixtures thereof, wherein said repeating units comprise amino acidresidues selected from the group consisting of hydrophobic amino acidand glycine residues, wherein said repeating units exist in aconformation having a β-turn which comprises a polypentapeptide unit ofthe formula:

    --X.sup.1 --(IPGVG).sub.n --Y.sup.1 --

wherein

I is a peptide-forming residue of L-isoleucine;

P is a peptide-forming residue of L-proline;

G is a peptide-forming residue of glycine;

V is a peptide-forming residue of L-valine; and

wherein X¹ is PGVG, GVG, VG, G or a covalent bond;

Y¹ is IPGV, IPG, IP, I or a covalent bond; and n is an integer from 1 to200, or n is 0, with the proviso that X¹ and Y¹ together constitute arepeating pentapeptide unit, in an amount sufficient to adjust thedevelopment of elastomeric force of the bioelastomer to a predeterminedtemperature.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1

Carbon-13 NMR spectra at 25 MHz in dimethylsulfoxide for A. Ile¹ -PPPand B. PPP. These spectra demonstrate the replacement of Val¹ by Ile¹ ;particularly in the upfield region the replacements of the β and γcarbon resonances of one valine residue by the CH₂ and CH₃ resonances ofisoleucine are apparent. The lack of extraneous peaks indicates a goodlevel of purity and the similar chemical shifts of the other fourresidues indicate similar conformations in this solvent.

FIG. 2

A. Temperature profiles for coacervation for the Ile¹ -PPP showing thehigh concentration limit to have an onset of aggregation at about 8° C.and a midpoint at 9° C. On dilution the profiles shift to highertemperatures. The polypentapeptide of elastin (PPP) profiles are givenfor comparison as the dashed curves. The addition of a CH₂ moiety causesa shift by 16° C. to lower temperatures for the coacervation process.

B. Ellipticity data at 197 nm for 0.025 mg Ile¹ -PPP per ml given as thesolid curve. The decrease in magnitude of the negative 197 nm bandindicates an increase in intramolecular order on increasing thetemperature, i.e., an inverse temperature transition. The dashed curveis the same data for 2.3 mg PPP per ml. The replacement of Val¹ by Ile¹shifts the transition 15° C. or more to lower temperatures.

C. Thermoelasticity data (temperature dependence of elastomeric force)for 20 Mrad γ-irradiation cross-linked Ile¹ -PPP coacervate shown as thesolid curve. There is a dramatic increase in elastomeric force thatcorrelates with the transition characterized in A and B above. Similardata for 20 Mrad cross-linked PPP coacervate are plotted on theright-hand ordinate as the dashed curve. The difference in scales is dueto the smaller cross-sectional area and a 40% extension for cross-linkedIle¹ -PPP whereas a larger cross-sectional area and a 60% extension wereused for cross-linked PPP. The elastic moduli are similar for the twoelastomers. Comparing the data in parts A, B, and C, it is apparent thatthe increased hydrophobicity of Ile over Val causes the inversetemperature transition to occur at lower temperatures and that theelastomeric force development occurs as a result of increasedintramolecular order.

FIG. 3

Circular dichroism spectra for 0.025 mg Ile¹ -PPP per ml of water at 2°C. (curve a) before the transition seen in FIG. 2B and at 35° C. (curveb) after the transition. As the large negative band near 195 nm isindicative of decreased polypentapeptide order, the decreased magnitudeof the large negative band on increasing the temperature is indicativeof increased order on raising the temperature. The spectrum at elevatedtemperature is indicative of Type II β-turn formation. For comparison isdata for PPP at 0.023 mg/ml at 15° C. before and 47° C. after thetransition shown in FIG. 2C. It is clear that Ile¹ -PPP and PPP have thesame conformation.

FIG. 4

Molecular structure proposed for the polypentapeptide of elastin (PPP).

A. The Type II Pro² -Gly³ β-turn as confirmed by the crystal structureof the cyclic conformational correlate.

B. Schematic representation of a helix with dimensions of the PPPβ-spiral.

C. Schematic representation of the PPP β-spiral showing the β-turns tofunction as spacers between turns of the spiral.

D. Detailed stereo pair of the axis view of the PPP β-spiral showingspace within the spiral for water and showing the Val⁴ -Gly⁵ -Val¹suspended segment.

E. Stereo pair side view of the β-spiral of the PPP showing the β-turnsfunctioning as spacers between turns of the spiral, showing open spaceson the surface of the β-spiral wherein intraspiral and extraspiral watercan exchange, and showing the suspended segment, Val⁴ -Gly⁵ -Val¹.

FIG. 5

Carbon-13 nuclear magnetic resonance spectrum at 25 MHz indimethylsulfoxide of the polytetrapeptide of elastin prepared bypolymerization of the GGVP permutation. All the carbon resonances areobserved with the correct chemical shifts and there are no extraneouspeaks.

FIG. 6

Temperature profiles for coacervation of the polytetrapeptide (PTP) ofelastin for a series of concentrations (the solid curves). Forcomparison are data for the polypentapeptide (PPP) of elastin (dashedcurves). The decreased hydrophobicity of the tetramer (VPGG) whencompared to the pentamer (VPGVG) results in a shift to highertemperature by some 25° C. for the aggregational process leading tocoacervate formation.

FIG. 7

Circular dichroism spectra for the PTP of elastin (solid curves) at lowtemperature 40° C. (curve a) and at elevated temperature 65° (curve b).The structural transition giving rise to the difference in the 195-200nm range is characterized as a function of temperature in FIG. 4A.Plotted for comparison is data for the PPP (dashed curves) before andafter the transition shown in FIG. 4A.

FIG. 8

A. Ellipticity at 200 nm as a function of temperature of the PTP ofelastin (solid curve) plotted on left-hand ordinate. For comparison arethe data for PPP (dashed curve) plotted on the right-hand ordinate. Thestructural transition is seen to occur for the PTP at a highertemperature by 20° to 25° C. The center of the transition correspondswith the aggregational process in FIG. 2, that is, the intramolecularconformational change precedes the association giving rise tocoacervation. As seen on comparison with part B, the characterization ofthe intramolecular structural transition by [θ]₂₀₀ closely parallels thedevelopment of elastomeric force.

B. Thermoelasticity data (temperature dependence of elastomeric force)for the 20 Mrad cross-linked PTP (solid curve) plotted on left-handordinate and for comparison for the 20 Mrad cross-linked PPP (dashedcurve)plotted on right-hand ordinate. The transition in elastomericforce is seen to correspond to the inverse temperature transition inintramolecular order as characterized by ellipticity in A above.

FIG. 9

Scale representations of the hydrophobicities of the repeating units(IPGVG), (VPGVG) and (VGPP) shown along with the midpoint temperaturesof the transitions as approximated from the ellipticity data ([θ]transition midpoint) and by the elastomeric force data (f transitionmidpoint) of FIGS. 2 and 8. This demonstrates that as the hydrophobicityof the repeating unit decreases the temperature for the transitionshifts proportionately to higher temperatures. As an inverse temperaturetransition in water is due to hydrophobic interactions, this verifiesthat the transition temperature correlates very closely withhydrophobicity of the repeating unit.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As noted above, elastin is comprised of a single protein. The sequenceof elastin can be described as a serial alignment of alanine-rich,lysine-containing cross-linking sequences alternating with glycine-richhydrophobic sequences. With more than 80% of the sequence known, themost striking hydrophobic sequences, both from the standpoint of lengthand of composition, are one that contains a polypentapeptide (PPP) andone that contains a polyhexapeptide (PHP). Elastin also contains arepeating polytetrapeptide (PTP). As a result of work conducted by thepresent inventors, the polypentapeptide of elastin when cross-linked hasbeen found to be elastomeric and the polyhexapeptide thereof has beenfound to be non-elastomeric and appears to provide a means for aligningand interlocking the chains during elastogenesis. From the present work,it has now also been found that the elastin polypentapeptide andpolytetrapeptide are both conformation-based elastomers that developentropic elasticity on undergoing an inverse temperature transition toform a regular β-turn containing dynamic structure.

A typical biological elastic fiber is comprised of a large elastin corecovered with a fine surface layer of microfibrillar protein. As notedpreviously, elastin is formed upon cross-linking of the lysine residuesof tropoelastin. The repeating elastin pentapeptide has the formula(VPGVG)_(n), while the repeating hexapeptide has the formula(VAPGVG)_(n), where n varies depending upon the species. The repeatingpolytetrapeptide unit has the formula (VPGG)_(n). These sequences, ofcourse, utilize the standard one-letter abbreviation for the constituentamino acids.

It has been found that these polypeptides are soluble in water below 25°C., but on raising the temperature they associate in thepolypentapeptide (PPP) and polytetrapeptide (PTP) cases, reversibly toform a viscoelastic phase, and in the polyhexapeptide (PHP) case,irreversibly to form a precipitate. On cross-linking, the former (PPP)and (PTP) have been found to be elastomers.

In part, the present invention resides in the dsscovery that attemperatures above 25° C. in water, PTP and PPP exhibit aggregation andform a water-containing viscoelastic phase, which upon cross-linking byγ-irradiation forms an elastomer. By contrast, PHP forms a granularprecipitate, which is not elastomeric. In fact, it has been found thatfor potential elastomers, such aggregation is readily reversible,whereas for non-elastomeric samples, such as PHP, temperature-drivenaggregation is irreversible and redissolution usually requires theaddition of trifluoroethanol to the aggregate.

For purposes of clarification, it is noted that the reversibletemperature elicited aggregation, which gives rise upon standing to adense viscoelastic phase, is called coacervation. The viscoelastic phaseis called the coacervate, and the solution above the coacervate isreferred to as the equilibrium solution.

Most importantly, however, in accordance with the present invention, ithas now been found that cross-linked PPP, PTP and analogs thereofexhibit elastomeric force development at different temperatures spanninga range of up to about 75° C. depending upon several controllablevariables. Moreover, it has been found for these cross-linked elastomersthat development of near maximum elastomeric force can occur over a verynarrow temperature range. Thus, by synthesizing bioelastomeric materialshaving varying molar amounts of the constituent pentamers and tetramerstogether with such units modified by hexameric repeating units, and bychoosing a particular solvent to support the initial viscoelastic phase,it is now possible to rigorously control the temperature at which theobtained bioelastomer develops elastomeric force.

In general, it has been found that the process of raising thetemperature to form the above elastomeric state is an inversetemperature transition resulting in the development of a regularnon-random structure, unlike typical rubbers, which utilizes, as acharacteristic component, hydrophobic intramolecular interactions. Theregular structure is proposed to be a β-spiral, a loose water-containinghelical structure with β-turns as spacers between turns of the helixwhich provides hydrophobic contacts between helical turns and hassuspended peptide segments. These peptide segments are free to undergolarge amplitude, low frequency rocking motions called librations.Consequently, a new mechanism of elasticity has now been developedcalled the librational entropy mechanism of elasticity.

It has now been found that the elastomeric force of these variousbioelastomers develops as the regular structure thereof develops.Further, it has been found that a loss of regular structure by hightemperature denaturation, results in loss of elastomeric force.Interestingly, this situation is just the reverse of that for therandom-chain-network theory of elasticity, in which the more nearlyrandom the polypentapeptide, the less the elastomeric force, and themore developed the β-turn containing structure, the greater theelastomeric force.

In the broadest sense, the present invention pertains to the developmentof a new entropy-based mechanism of elasticity. The mechanism thereforappears to be derived from a new class of polypeptide conformationscalled β-spirals wherein β-turns recur with regularity in a loosewater-containing helix. The β-spiral is the result of intramolecularinterturn hydrophobic interactions which form on raising the temperaturein water. In the β-spiral of the elastomeric polypentapeptide ofelastin, (Val¹ -Pro² -Gly³ -Val⁴ -Gly⁵)_(n), the type II Pro² -Gly³β-turns function as spacers, with hydrophobic contacts, between theturns of the helix, which results in the segments of Val⁴ -Gly⁵ -Val¹being suspended. Being essentially surrounded by water, the peptidemoieties of the suspended segments are free to undergo large rockingmotions referred to as librations which become damped on stretching. Thedecrease in amplitude of librations on stretching constitutes a decreasein entropy and it appears that the decrease in free energy due to theincrease in entropy on returning to the relaxed state is the drivingforce for elastomeric retraction.

In accordance with the present invention, upon raising the temperatureof the polypeptide-solvent system, such as PPP-water, for example, thehydrophobic side chains such as those of Pro and Val when dispersed inwater are surrounded by water having a clathratelike structure, that is,by water that is more ordered than normal bulk water. Upon raising thetemperature, an amount of this more ordered clathrate-like watersurrounding the hydrophobic groups becomes less ordered bulk water asthe hydrophobic chains associate to form a more ordered polypeptide. Itappears that it is the optimization of intramolecular hydrophobiccontact that assists the polypeptide in wrapping up into a loose helix.Adherence to the Second Law of Thermodynamics appears to be maintainedby the requirement that the decrease in entropy of the polypeptideportion of the system be less than the increase in entropy of the waterin the system. Since ΔG=0 at the temperature midpoint (T_(mp)) of astructural transition between a pair of states, then T_(mp) =ΔH/ΔS. Ifthe entropy change, ΔS, derives from the hydrophobicity of the repeatingunit, as it would in the clathrate-like water mechanism, than anincrease in the hydrophobicity of the repeating unit can be used toexplain the decrease in T_(mp), the midpoint of the inverse temperaturetransition. In fact, in accordance with the present invention, it hasbeen found that a decrease in the hydrophobicity of the repeating unitresults in an increase in T_(mp). Conversely, an increase in thehydrophobicity of the repeating units results in a decrease in T_(mp).

The above principle can be demonstrated by substituting the morehydrophobic isoleucine (Ile) for valine (Val) in the elastinpolypentapeptide, (Ile¹ -Pro² -Gly³ -Val⁴ -Gly⁵)_(n), i.e., Ile¹ -PPP,to produce a substituted polypentapeptide which has properties similarto PPP, except that the described transition occurs at a lowertemperature. See FIGS. 1-3.

For purposes of clarity, it is noted that for the above numberedsequence and all sequences hereafter, the superscript numbering systemis a sequence numbering based upon the dominant secondary structuralfeature of these repeating sequences which is the type II Pro² -Gly³β-turn, a ten atom hydrogen bonded ring involving the C=0 of residue 1and the NH of residue 4.

The present invention also has been found to extend to thepolytetrapeptide of elastin. It is recalled that this repeating unit hasthe formula (Val¹ -Pro² -Gly³ -Gly⁴)_(n), which also forms a β-spiralsimilar to PPP. However, the temperature of aggregation for PTP occursat a higher temperature than for PPP. In essence, for both thepolypentapeptide and polytetrapeptide repeating units of elastin, thepresent inventors have found that the temperature of the transition forthe development of elastomeric force is proportional to thehydrophobicity of the repeating unit. This is shown graphically in FIG.9. Hence, two important principles elucidated by the present inventionmay now be stated. First, is that elastomeric force development occursdue to an inverse temperature transition resulting in increasedpolypeptide order by raising the temperature. Secondly, the temperatureof this transition for the development of elastomeric force isproportional to the hydrophobicity of the repeating unit in thebioelastomer.

In accordance with the present invention, analogs of both the elastinpolypentapeptide PPP and the polytetrapeptide (PTP) and combinationsthereof are contemplated. For example, it has been found that thetemperature of transition for Ile¹ -PPP shifts to a lower temperature byan amount calculable from the increase in hydrophibicity relative to PPPusing the hydrophobicity scales shown in FIG. 9. Thus, by carefullychoosing a new analog with a different repeating unit hydrophobicity,the transition temperature for the development of elastomeric force canbe predictably shifted to a different temperature. In fact, byjudiciously selecting various repeating units and combinations thereof,along with various solvent mixtures it is now possible to select atransition temperature from within a range of up to about 75° C., fromabout -25° C. to about +50° C.

As noted previously, the most striking repeating sequence of the elastinpolypentapeptide is (Val¹ -Pro² -Gly³ -Val⁴ -Gly⁵)_(n), wherein, forexample, n is 13 for chicks and 11 for pigs. The polypentapeptide issoluble in water at all proportions below 25° C. On raising thetemperature above 25° C., aggregation occurs and the aggregate settlesto form a dense viscoelastic phase coacervate that at 40° C. is about38% peptide and 62% water by weight. The process of PPP coacervation, asnoted, is entirely reversible. Moreover, on cross-linking, the PPPcoacervate is found to be elastomeric. The coacervate concentration ofPPP as well as the elastomeric γ-irradiation cross-linked PPP coacervateundergo an inverse temperature transition, which commences at 25° C. andwhich reaches completion near 37° C. Over the same temperature range,the elastomeric force of the cross-linked PPP coacervate increasesdramatically from near zero at 20° C. to full force near 40° C. Above40° C., the elastomeric force divided by the temperature (°K) becomesquite constant.

This indicates that the cross-linked PPP is a dominantly entropicelastomer. That is, the entropic component of the elastomeric forcedepends upon the decrease in numbers of low energy states accessible tothe polymer on extension, whereas the internal energy component ofelastomeric force results from stressing of bonds which would increasethe probability of rupture of the elastomer. Interestingly enough, withthe development of near maximum entropic elastomeric force upon raisingthe temperature from 25° C. to 37° C., it would appear that thepolypentapeptide of elastin specifically evolved for warm-bloodedanimals. Further, it appears that this evolution occured at a relativelyearly stage in mammalian evolution, inasmuch as these repeating peptidesequences appear to have remained unchanged throughout the past 200million years of mammalian evolution.

Thus, in part, the present invention is predicated upon the finding thatit is possible to change the temperature of transition by modifying thePPP. In particular, it has been found that by increasing thehydrophobicity of the PPP repeating unit, the viscoelastic phasetransition occurs at lower temperatures, while by decreasing thehydrophobicity of the repeating unit, this transition occurs at highertemperatures. Of course, when modifying the hydrophobicity, it isnecessary to do so in a way such that elasticity is retained.

For example, modifications of the repeating pentamers have been madewhich destroy the molecular structure required for elasticity, such asthe Ala¹ and Ala⁵ analogs. The Ala¹ and Ala⁵ analogs, the formerdecreasing and the latter increasing pentamer hydrophobicity, result inthe formation of granular precipitates on raising the temperature ofaqueous solutions rather than forming viscoelastic coacervates andγ-irradiation cross-linking of the Ala⁵ -PPP precipitate results in ahard material that simply breaks upon stretching. In accordance with thepresent discovery, it is believed that these analogs fail to produceelastomeric polymers for different but consistent reasons. First, theAla¹ analog does not appear to aIlow for important Val¹ γCH₃ . . . Pro²δCH₂ intramolecular hydrophobic contacts required to form a viscoelasticcoacervate. The Ala⁵ analog appears to interfere with librationalmotions in the Val⁴ -Gly⁵ -Val¹ suspended segment of the proposed PPPmolecular structure. As noted, the librations are central to theproposed librational entropy mechanism of elasticity.

By contrast, the hydrophobicity of the repeating pentamer can be easilyincreased by introducing a--CH₂ --moiety, for example, in residue 1while maintaining β-branching, that is, to utilize the Ile¹ analog ofPPP, i.e., (Ile¹ -Pro² -Gly³ -Val⁴ -Gly⁵)_(n). With a greater than50,000 molecular weight, Ile¹ -PPP reversibly forms a viscoelasticcoacervate with the onset of coacervation being at 8° C. rather than 24°C. as for unsubstituted PPP. It appears from circular dichroism datathat Ile¹ -PPP and PPP have identical conformations both before andafter the transitions and that the transition to increasedintramolecular order on increasing the temperature is also shifted by15° C. or more to lower temperatures. Further, the dramatic increase inelastomeric force on raising the temperature of the γ-irradiationcross-linked coacervate is similarly shifted to a lower temperature forthe Ile¹ -PPP analog. Thus, with this analog, a coupling of temperaturedependent elastomeric force development and molecular structure isdemonstrated. This, of course, means that it is now possible torationally design polypeptide elastomers that undergo transitions atdifferent temperatures and that would function as entropic elastomers indifferent temperature ranges.

As noted above, by increasing the hydrophobicity of PPP, such as bysubstituting Ile¹ for Val¹ in the pentameric sequence of --(VPGVG)_(n)to form --(IPGVG)_(n), it is now possible to accomplish at least twodistinct objectives.

First, it is now possible to prepare, for example, the "homopolymeric"polypentapeptide of --(IPGVG)_(n) --, i.e., Ile¹ -PPP, which, as noteddissolves in water at 4° C., and upon raising the temperature to 8° C.,exhibits aggregation. After cross-linking the coacervate byγ-irradiation, it is observed that essentially full elastomeric force isexhibited at about 25° C. for the cross-linked Ile¹ -PPP as opposed tothe 40° C. temperature required for the unsubstituted PPP. Thus, thetemperature ordered transition for Ile¹ -PPP occurs at a temperatureapproximately 15° C. lower than for PPP.

Secondly, it is now also possible to prepare mixed "copolymers", forexample, of the polypentapeptides --X¹ --(IPGVG)_(n) --Y¹ --and --X²--(VPGVG--)_(n) --Y² --which exhibit variable and controllabletransition temperatures which are in between the separate transitiontemperatures of PPP and Ile¹ -PPP. Further, a great degree of control ispossible inasmuch as the transition temperature obtained is directlyproportional to the molar ratios of the respective pentapeptidesincorporated therein.

Perhaps the most striking feature of the increased hydrophobicity PPPcross-linked analogs is that nearly full elastomeric force can bereached over a very narrow temperature range. For example, forcross-linked Ile¹ -PPP, it is found that the elastomeric force thereofshows an abrupt increase from essentially zero at 8° C. tothree-quarters of full force at 10° C., and essentially full force by20°-25° C. Such an increase in elastomeric force over only a 2° C.temperature differential is, indeed, unprecedented and can be controlledby the percent extension in relation to swelling of the elastomer onlowering the temperature.

Although Ile¹ -PPP is an excellent example of an increasedhydrophobicity PPP analog, any PPP analog, which reduces thehydrophobicity of the repeating pentameric unit, while retaining theelasticity of the polypeptide, and without interfering with either theformation of the viscoelastic coacervate or the librational motion iswithin the ambit of the present invention.

For example, in addition to repeating unit sequences of --IPGVG--_(n),using Ile¹, it is also possible to effect a variety of othersubstitutions. In general, a pentapeptide repeating unit of the formula:

    --(R.sub.1 PR.sub.2 R.sub.3 G).sub.n --

is within the ambit of the present invention, wherein R₁ is selectedfrom the group consisting of Phe, Leu, Ile, and Val; R₂ is selected fromthe group consisting of Ala and Gly; R₃ is selected from the groupconsisting of Phe, Leu, Ile, and Val; and n is an integer from 1 to 200;and P is L-proline and G is glycine.

Notably, the above substitutions modify the hydrophobicity of therepeating unit so as to attenuate the transition temperature for nearmaximum elastomeric force development, of course, without destroying theelasticity of the bioelastomer.

In the above formula, it is noted that the amino acid Leu is, of course,Leucine. R₁, R₂ and R₃ correspond to positions 1, 3 and 4 in thenumbered sequence as described herein.

Interestingly, with Phe¹ -PPP in water, it is possible to shift thetemperature of transition initiation from 25° C. for PPP to about 0° C.Furthermore, this shift can be driven to even lower temperatures byutilizing mixed solvent systems of water/ethylene glycol orwater/dimethyl sulfoxide (DMSO). For example, by using the Phe¹-PPP/water-ethylene glycol system, a transition temperature of as low asabout -25° C. can be obtained. Of course, a range of transitiontemperatures can be obtained between 0° C. and about -25° C. for thePhe¹ -PPP/water-ethylene upon glycol system depending upon the amount ofethylene glycol added. It has been found that very low transitiontemperatures are obtained using approximately 50/50 mixtures ofwater/ethylene glycol.

Conversely, the maximum shift to higher transition temperatures islimited by the denaturation of the polypeptide. With the presentelastomeric polypeptides, this upper limit appears to be about 50° C.,with denaturation beginning above 60° C.

However, as noted previously, the present invention includes not onlyPPP analogs, such as Ile¹ -PPP, Phe¹ -PPP or Ala³ -PPP but all PPPanalogs, and bioelastomers containing the same, which have transitiontemperatures, and, hence, temperatures of near maximum elastomeric forcedevelopment, which are different from PPP; while retaining elasticity.Given, the present disclosure, one skilled in the art could clearlyascertain additional PPP analogs, and bioelastomers incorporating thesame which meet the above criteria.

As noted above, the increased hydrophobicity analog, such as Ile¹ -PPPmay be synthesized as a "homopolymer", or a "copolymer" of --X²--(VPGVG--)_(n) --Y² --and --X¹ --(IPGVG--)_(n) --Y¹ --may besynthesized with the molar ratio of the constituent pentamers beingdependent upon the desired temperature for elastomeric forcedevelopment. However, in general, in such "copolymers", the --X¹--(IPGVG--)_(n) --Y¹ --pentameric component is present in about 1-99% ofthe total pentameric molar content, while the --X² --(VPGVG--)_(n) --Y²--pentameric component is present in about 99-1% of the total pentamericmolar content. More preferably, the --X¹ --(IPGVG--)_(n) --Y¹--component is present in about 5-95% of the total pentameric molarcontent, while the --X² --(VPGVG--)_(n) --Y² --component is present inabout 95-5% of the total pentameric molar content. However, anycombination of relative molar amounts can be used as dictated by thedesired transition temperature.

Thus, in accordance with one aspect of the present invention,bioelastomers can be prepared which contain elastomeric units comprisingtetrapeptide, or pentapeptide or units thereof modified by hexapeptiderepeating units and mixtures thereof, wherein said repeating unitscomprise amino acid residues selected from the group consisting ofhydrophobic amino acid and glycine residues, wherein the repeating unitsexist in a conformation having a β-turn which comprises apolypentapeptide unit of the formula:

    --X.sup.1 --(IPGVG--).sub.n --Y.sup.1 --

wherein

I is a peptide-forming residue of L-isoleucine;

P is a peptide-forming residue of L-proline;

G is a peptide-forming residue of glycine;

V is a peptide-forming residue of L-valine; and

wherein X is PGVG, GVG, VG, G or a covalent bond; Y is IPGV, IPG, IP orI or a covalent bond; and n in both formulas is an integer from 1 to200; or n is 0, with the proviso that X¹ and Y¹ together constitute arepeating pentapeptide unit, in an amount sufficient to adjust thedevelopment of elastomeric force of the bioelastomer to a predeterminedtemperature.

However, the present invention also relates, as noted above, tobioelastomers which contain elastomeric units comprising tetrapeptide,or pentapeptide or units thereof modified by hexapeptide repeating unitsand mixtures thereof, wherein said repeating units comprise amino acidresidues selected from the group consisting of hydrophobic amino acidand glycine residues, wherein the repeating units exist in aconformation having a β-turn which comprises (A) a polypentapeptide unitof the formula:

    --X.sup.1 --(IPGVG--).sub.n --Y.sup.1 --

and (B) a polypentapeptide unit of the formula:

    --X.sup.2 --(VPGVG--).sub.n --Y.sup.2 --

wherein for the above formulas,

I is a peptide-forming residue of L-isoleucine;

P is a peptide-forming residue of L-proline;

G is a peptide-forming residue of glycine;

V is a peptide-forming residue of L-valine; and

wherein X¹ and X² are each PGVG, GVG, VG, G or a covalent bond; Y¹ isIPGV, IPG, IP or I or a covalent bond; Y² is VPGV, VPG, VP, V or acovalent bond; and n in both formulas an integer from 1 to 200; or n inboth formulas is 0, with the proviso that X¹ and Y¹ together, and X² andY² together constitute a repeating pentapeptide unit, in relativeamounts sufficient to adjust the development of elastomeric force of thebioelastomer to a predetermined temperature.

It should be noted that bioelastomeric polypeptide chains containingeither one or both of the above pentapeptide repeating units can besynthesized using any of the pentapeptide "monomers" that arepermutations of the basic sequence. However, if the polymer is notsynthesized using the pentapeptide "monomers", but rather is synthesizedby sequential adding of amino acids to a growing peptide, such as in thecase of an automatic peptide synthesizer, the designation of therepeating unit is somewhat arbitrary. For example, the peptideH-V(PGVGVPGVGVPGVGVPGVGV)P-OH can be considered to consist of any of thefollowing repeating units and end groups: H-(VPGVG)₄ -VP-OH,H-V-(PGVGV)₄ -P-OH, H-VP-(GVGVP)₄ -OH, H-VPG-(VGVPG)₃ -VGVP-OH, orH-VPGV-(GVPGV)₃ -GVP-OH, for example.

Furthermore, it is entirely possible and within the ambit of the preseninvention that mixed repeating units such as those of the formula--VPGVGIPGVG--_(n) can be incorporated into the bioelastomers of thepresent invention.

Synthesis of the elasticity promoting and modifying segments, which areincorporated into the final elastomeric polypeptide, is straightforwardand easily accomplished by a peptide chemist. The resulting intermediatepeptides generally have the structure, B¹ -(repeating unit)_(n) -B²,where B¹ and B² represent any chemically compatible end group on theamino and carboxyl ends of the molecule, respectively, and n is aninteger of from 2 to about 200. Of course, when B¹ is --H and B² is--OH, and n is 1, the compound is either the pentapeptide H-VPGVG-OH orH-IPGVG-OH. When n is greater than 1, the compound intermediate is apolypentapeptide. The same will hold true when utilizing tetramericrepeating units in the present bioelastomers.

It should be noted that the term "hydrophobic amino acid" refers toamino acids which have appreciably hydrophobic R groups as measured on ahydrophobicity scale generated by measuring the relative solubilities ofthe amino acids in organic solvents. In this respect, see Arch. Biochem.Biophy, Bull and Breese, Vol. 161, 665-670 (1974). By this method, allamino acids which are more hydrophobic than glycine may be used. Morespecifically, preferable hydrophobic amino acids are Ala, Val, Leu, Ileand Pro.

It should also be noted that it is entirely possible that one or moreamino acid residues or segments of amino acid residues not present inthe normal pentapeptide or tetrapeptide sequence may be interspersedwithin a polypentapeptide or polytetrapeptide portion of an elastomericpolypeptide chain.

The bioelastomers of the present invention, regardless of the particularfunctional repeating unit incorporated therein, may have these repeatingunits incorporated either in the form of block or random copolymers aslong as the desired shift in temperature of elastomeric forcedevelopment of the bioelastomer is obtained. As noted above, byconsidering the transition temperatures and temperatures of elastomericforce development for two PPP or PTP analogs, or even for a PPP analogand a PTP analog, it is possible to attain a desired intermediatetransition temperature and temperature of elastomeric force developmentby directly correlating the molar ratios of each analog componenttherewith. For example, a 50/50 molar ratio of two analog componentswould give rise to a bioelastomer "copolymer" having a transitiontemperature and temperature of elastomeric force developmentapproximately in between those of the analog components.

Additionally, it is also noted that the elastomeric units used inconjunction with all aspects of the present invention, i.e., whether therepeating unit is PPP, PTP or analogs thereof, may also comprise thosedescribed in U.S. Pat. Nos. 4,187,852; 4,474,851; 4,500,700, and4,589,882 and U.S. patent applications 533,670, 793,225 and 853,212 allof which patents and patent applications are incorporated herein intheir entirety.

The aspect of the present invention with respect to PPP and analogsthereof will now be illustrated by Examples, which are provided only forthe purpose of illustration and are not intended to limit the presentinvention.

EXAMPLES Peptide Synthesis

The synthesis of Ile¹ -PPP was carried out by the classical solutionmethods as shown in Scheme I.

In the following Examples, the following abbreviations will be used:Boc, tert-butyloxycarbonyl; Bzl, benzyl; DMF, dimethylformamide; DMSO,dimethylsulfoxide; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;HOBt, 1-hydroxybenzotriazole; IBCF, isobutylchloroformate; NMM,N-methylmorpholine; ONp, p-nitrophenbylester; TFA, trifluoroacetic acid;PPP, (VPGVG)_(n) ; Ile¹ -PPP, (IPGVG)_(n) ; V, valine; I, isoleucine; P,proline; G, glycine. ##STR1##

The sequence of the starting pentamer for polymerization is preferablyGly-Val-Gly-Ile-Pro rather than Ile-Pro-Gly-Val-Gly, because thepermutation with Pro as the C-terminal amino acid produces highmolecular weight polymers in better yields. The approach to thesynthesis entailed coupling the tripeptide Boc-GVG-OH (II) withH-IP-OBzl, each in turn being synthesized by the mixed anhydridemethodology of J. R. Vaughan et al, J. Am. Chem. Soc., 89, 5012 (1967).The possible formation of the urethane as a by-product during thereaction of Boc-Ile-OH with H-Pro-OBzl by the mixed anhydride method wasavoided by carrying out the reaction in the presence of HOBt. Thedipeptide was also prepared using EDCI for confirmation of the product.The pentapeptide benzylester (III) was hydrogenated to the free acid(IV) which was further converted to the p-nitrophenylester (V) onreacting with bis(p-nitrophenyl)carbonate. On removing the Boc-group, aone molar solution of the active ester in DMSO was polymerized in thepresence of 1.6 equiv. of NMM. The polypeptide was dialyzed againstwater using a 50,000 dalton cut-off dialysis tubing and lyophilized. Thepurity of the intermediate and final products was checked by carbon-13nuclear magnetic resonance, elemental analyses and thin layerchromatography (TLC).

Elemental analyses were carried out by Mic Anal, Tuscon, Ariz. All aminoacids are of L-configuration except for glycine. Boc-amino acids werepurchased from Bachem, Inc., Torrance, Calif. HOBt was obtained fromAldrich Chemical Co., Milwaukee, Wis. TLC was performed on silica gelplates purchased from Whatman, Inc., Clifton, N.J. in the followingsolvent systems: R_(f) ¹, CHCl₃ (C):CH₃ OH(M):CH₃ COOH(A), 95:5:3; R_(f)², CMA (85:15:3); R_(f) ³, CMA (75:25:3); R_(f) ⁴, CM (5:1). Meltingpoints were determined with a Thomas Hoover melting point apparatus andare uncorrected.

Boc-Ile-Pro-OBzl (mixed anhydride method) (I): Boc-Ile-OH (12.01 g, 0.05mole) in DMF (50 ml) was cooled to 0° C. and NMM (5.49 ml) was added.After cooling the solution to -15° C. isobutylchloroformate (6.48 ml)was added slowly while maintaining the temperature at -15° C. andstirred for 10 minutes at which time HOBt (7.65 g) was added andstirring was continued for additional 10 minutes. A pre-cooled solutionof HCl-H-Pro-OBzl (12.09 g, 0.05 mole) in DMF (50 ml) and NMM (5.49 ml)was added to the above solution and the completeness of the reaction wasfollowed by TLC. The reaction mixture was poured into a cold saturatedNaHCO₃ solution and stirred for one hour. The peptide was extracted intoCHCl₃ and washed with acid and base (0.5 N NaOH to remove HOBt), and onevaporating the solvent the product was obtained as an oil in 92% yield.R_(f) ¹, 0.65. Anal. Calcd. for C₂₃ H₃₄ N₂ O₅ : C 66.00, H 9.19, N6.69%. Found: C 65.58, H 8.28, N 7.13%.

Boc-Ile-Pro-OBzl (using EDCI): Boc-Ile-OH (7.20 g, 0.03 mole) and HOBt(5.05 g, 0.033 mole) in DMF (30 ml) was cooled to -15° C. and EDCI (6.32g, 0.033 mole) was added. After stirring for 20 minutes, a pre-cooledsolution of HCl-H-Pro-OBzl (7.25 g, 0103 mole) in DMF (30 ml) and NMM(3.3 ml) was added and stirred overnight at room temperature. Afterevaporating DMF, the residue was taken into CHCl₃ and extracted with 20%citric acid and 0.5N NaOH. The solvent was removed and the product wasobtained as an oil in almost quantitative yield which was identical tothe product obtained by the mixed anhydride method.

Boc-Gly-Val-Gly-Ile-Pro-OBzl (III): Boc-GVG-OH (II) (20) (5.6 g, 0.017mole) was coupled with H-Ile-Pro-OBzl (6.7 g, 0.019 mole) (obtained bydeblocking I with HCl/Dioxane) in the presence of EDCI (3.65 g, 0.019mole) and HOBt (2.9 g, 0.019 mole) and the product was worked up asdescribed above to obtain 8.8 g of III (yield: 82.4%), m.p. 107°-108° C.(decomp.) R_(f) ¹, 0.44; R_(f) ², 0.75. Anal. calcd. for C₃₂ H₄₉ N₅ O₁₀: C 60.83, H 7.81, N 11.08%. Found: C 61.12, H 8.06, N 11.06%.

Boc-Gly-Val-Gly-Ile-Pro-OH (IV): III (7.8 g, 0.0123 mole) was taken inacetic acid (80 ml) and hydrogenated in the presence of 10% Pd-C (1 g)at 40 psi. After filtering the catalyst with the aid of celite, thesolvent was removed under reduced pressure, triturated with ether,filtered, washed with ether then pet. ether and dried to obtain 6.5 g ofthe product (yield: 97.3%), m.p. shrinks at 127° C. and decomp. at 145°C. R_(f) ³, 0.24; R_(f) ⁴, 0.11. Anal. Calcd. for C₂₅ H₄₃ N₅ O₁₀.1/2 H₂O: C 54.52, H 8.05, N 12.71%. Found: C 54.32, H 8.02, N 12.59%.

Boc-Gly-Val-Gly-Ile-Pro-ONp (V): IV (5.41 g, 0.01 mole) in pyridine (40ml) was reacted with bis(p-nitrophenyl)carbonate (4.56 g, 0.015 mole)following the completeness of the reaction by TLC. Pyridine was removed;the residue was taken into CHCl₃ and extracted with acid and base. Thep-nitrophenyl ester obtained was chromatographed over a silica gel(200-400 mesh) column. After initial washing with CHCl₃, 4.8 g of V wasobtained when eluted with 35% acetone in CHCl₃ (yield: 71.4%), m.p.97°-100° C. R_(f) ², 0.72; R_(f) ⁴, 0.75; Anal. Calcd. for C₃₁ H₄₆ N₆O₁₂.2H₂ O: C 53.28, H 7.21, N 12.02%. Found: C 53.76, H 6.83, N 12.01%.

H-(Gly-Val-Gly-Ile-Pro)_(n) -OH(VI): The Boc-group was removed from V(3.8 g, 0.0057 mole) by reacting with TFA (35 ml) for 45 min. TFA wasremoved under reduced pressure, triturated with ether, filtered, washedwith ether, pet. ether and dried. The TFA salt (3.3 g, 0.0049 mole) inDMSO (4.9 ml) was stirred for 14 days in the presence of NMM (0.86 ml,0.0078 mole). After diluting with water in the cold, the polypeptide wasdialyzed using a 50 kD cut-off dialysis tubing changing the water dailyfor 15 days. The retentate was lyophilized to obtain 1.81 g of the Ile¹-polypentapeptide (yield: 88%). The carbon-13 NMR spectrum is presentedin FIG. 1 along with that of the regular polypentapeptide forcomparison.

Temperature Profiles for Coacervation

The temperature dependence for aggregation of the polypentapeptide isfollowed as the development of turbidity at 300 nm using a Cary 14spectrophotometer. The sample cell is placed within a chamber vibratingat 300 Hz in order to facilitate equilibrium and to keep the aggregatesfrom settling. The scan rate is 30° C./hour and the temperature wascontrolled with a Neslab ETP-3 programmer and monitored with an Omega199A thermocouple monitor placed at the cell. The turbidity as afunction of temperature provides a temperature profile for coacervationwhich is found to be concentration dependent. As the concentration israised, the profile shifts to lower temperatures until further increasesin concentration cause no further lowering of the temperature foraggregation. This defines the high concentration limit. The temperaturefor the onset of coacervation at the high concentration limit coincideswith the temperature for the onset of the transition within thecoacervate itself, even when there is no appreciable change in watercontent of the coacervate. The temperature for the midpoint of thetemperature profile for the high concentration limit has been shown tocorrelate with the molecular weight of the polypentapeptide. When themidpoint is 25° C. for the PPP, the molecular weight is close to 100,000daltons as calibrated by dialysis. For the Ile¹ -PPP with a midpoint of9° C., the molecular weight is greater than 50,000 daltons, as thesynthetic polypeptide was retained by a 50,000 daltons dialysismembrane. The dialysis was carried out at 4° C. where the Ile¹ -PPP isin solution.

Circular Dichroism Measurements

The circular dichroism studies were carried out on a Cary 60spectropolarimeter equipped with a Model 6001 CD accessory modified for330 Hz modulation of the left and right circularly polarized light. Aconcentration of 0.025 mg Ile¹ -PPP/ml of doubly distilled water wascharacterized in a 10 mm path length cell. The low concentration wasused to keep the size of the aggregate sufficiently small as not tocause light scattering distortions of the CD spectra. Even at this lowconcentration with this more hydrophobic polypentapeptide, above 35° C.the size of the aggregates was sufficient to cause particulatedistortions as was apparent with the red shifting and dampening of thelong wavelength negative band. The temperature was controlled andmonitored from the cell as for the temperature profiles forcoacervation.

Formation of the Elastomeric Matrix

In preparation for γ-irradiation cross-linking (the means of forming theelastomeric matrix), 130 milligrams of peptide Ile¹ -PPP were dissolvedin 220 milligrams of water in a cryotube. The sample was then shearoriented at 0° C. in a previously described pestlecryotube arrangement.Gamma-irradiation was carried out at the Auburn University NuclearScience Center at a dose rate of approximately 8,000 Roentgen/min andfor sufficient time to achieve a 20×10⁶ radiation absorbed dose (20Mrad).

Thermoelasticity Studies

Thermoelasticity studies were carried out on a stress-stain instrumentbuilt in this Laboratory. The sample is mounted in two Delrin clamps.The top clamp is attached to a Statham UTC strain-gauge and the assemblyis fixed. The bottom clamp is attached to a moving platform driven by avariable speed motor. Both clamps are enclosed in a thermostated waterjacket. An inner chamber contains the solvent in which the elastomer isimmersed which in this case is doubly distilled water. The sample wasfixed in the top clamp and equilibrated in water at 60° C. for about anhour. The strain-gauge signal conditioner was balanced for zero forceand the bottom clamp was attached to the sample. The sample was left toset overnight at room temperature. The bottom clamp was then adjustedfor zero force and the distance between the clamps was measured. Theelastomer was elongated to 40% extension at 5° C. and elastomeric forcewas then determined as a function of temperature. Equilibrium time toachieve constant force at a given temperature was typically twenty-fourhours. Force measurements were made in 2° C. increments through thesharp rise in force and 5° C. increments at higher temperatures.

RESULTS Temperature Profiles for Coacervation

The Ile¹ -PPP can be dissolved in water on standing below 8° C. Onraising the temperature of the solution above 8° C., the solutionbecomes cloudy; on standing at the elevated temperature settling occursand a viscoelastic phase forms in the bottom of the vial; on placing thevial in an ice bath the cloudiness immediately clears and theviscoelastic phase readily dissolves. Thus the Ile¹ -PPP coaceravateswhen dissolved in water. The temperature profiles for coacervation(turbidity profiles) are shown in FIG. 2A for different concentrations.As the concentration is raised, the temperature profile shifts to lowertemperature. At 40 mg/ml, the high concentration limit (i.e, the lowerconcentration for which further increases in concentration cause nofurther lowering of the temperature for the onset of aggregation), themidpoint for the temperature profile for coacervation of Ile¹ -PPP is 9°C.

Included for comparison in FIG. 2A are the data for the PPP of elastindemonstrating the temperature profile midpoint for the highconcentration limit to be 25° C. The simple addition of a CH₂ moiety tothe 409 dalton repeating unit causes the onset of aggregation to shiftto lower temperatures by 16° C. Observing that curve f (0.1 mg Ile¹-PPP/ml) and curve k (1.0 mg PPP/ml) are comparable with respect to thehigh concentration limits for each high molecular weight polymersuggests that the size of the aggregate for Ile¹ -PPP is greater for agiven concentration than it is for a comparable concentration of PPP.This will be relevant to comparisons made in the circular dichroismdata.

Circular Dichroism

In FIG. 3 are the circular dichroism curves for Ile¹ -PPP in water(0.025 mg/ml) at 2° C. and at 35° C. The low concentration was chosen inorder that the size of the aggregate formed on association at 35° C.would have limited particulate distortions in the CD spectrum. At lowtemperature there is a large negative band near 195 nm. Such a negativeband is characteristic of disordered proteins and polypeptides, though astandard value for this negative peak for complete disorder is -4×10⁴rather than the observed value of -1.2×10⁴. Also the negative band near220 nm, rather than zero ellipticity or a positive band which are takenas indicative of complete disorder, suggests elements of order at lowtemperature. The decrease in intensity of the negative CD band near 195nm on raising the temperature of Ile¹ -PPP in water indicates anincrease in intramolecular order on raising the temperature, that is,there is an inverse temperature transition in an aqueous system. Thisindicates that hydrophobic interactions are developing as the orderedstate develops. The intramolecular increase in order begins just above0° C. and is complete by about 30° C. for a concentration of 0.025mg/ml. As is apparent from the data in FIG. 2A, the transition wouldhave been complete at a lower temperature (the transition would havebeen sharper) if the CD data could have been obtained at higherconcentration without significant particulate distortion. Shown forcomparison in FIG. 2B is the value of [θ]₁₉₇ as a function oftemperature for PPP in water (2.3 mg/ml) where the transition isobserved to be shifted to higher temperature by about 15° C. In FIG. 3again for comparison are the CD spectra for PPP (0.023 mg/ml) at 15° C.below the onset temperature for the transition and at 47° C. where thetransition is largely complete for this dilute concentration. It isapparent that Ile¹ -PPP and PPP have essentially identical conformationsbelow the onset temperature for the transition and that they haveessentially identical conformations after the transition is mostlycompleted. Thus while maintaining essentially identical conformations,which is assisted by the retention of β-branching, the addition of a CH₂moiety lowers the transition toward increased order by about 15° C.

Characterization of Elasticity

The elastic (Young's) modulus determined for 20 MRAD cross-linked Ile¹-PPP coacervate was 4×10⁵ dynes/cm² which is within the range of valuesobtained for 20 Mrad cross-linked PPP. The range of values is due tovariable vacuolization occurring during γ-irradiation which makesdifficult accurate measurement of cross-sectional area. It should beappreciated, however, that γ-irradiation causes no detectable polymerbreakdown when measured by carbon-13 and nitrogen-15 NMR.

The temperature dependence of elastomeric force is given in FIG. 2C foran elastomeric band of Ile¹ -PPP at 40% elongation. A near zeroelastomeric force is measured at 8° C.; on raising the temperature thereis a dramatic, an abrupt increase in elastomeric force. Full force isreached by 25° C. and becomes essentially constant with furtherincreases in temperature. Included in FIG. 2C, again for comparison, isthe data for 20 MRAD cross-linked PPP coacervate at 60% extension. Thereis similarly a dramatic rise in elastomeric force with increase intemperature but this curve is displaced about 15° C. to highertemperatures. Thus the results contained in FIG. 2 demonstrate withthree different physical methods that the addition of a CH₂ moiety (thereplacement of Val by Ile) shifts the transition to lower temperaturesby 15° C. without changing the conformation of the polypentapeptidebefore and after the transition. While the previously reported data onthe naturally occuring PPP of elastin demonstrate a correlation ofincreased structural order with increased elastomeric force, the Ile¹-PPP data with the transition shifted by 15° C. appear to confirm anobligatory coupling of increased order with increased elastomeric force.

In fact, the correlation of increased order with increased elastomericforce is seen with the PPP. When the transition is shifted to lowertemperatures, as in Ile¹ -PPP, the development of elastomeric forcefaithfully shifts to lower temperatures. There appears in suchelastomeric polypeptides to be a strict coupling between increasingorder and increasing elatomeric force; and the molecular structureprovides an understanding as to how this can occur. The similarconformations of PPP and Ile¹ -PPP (see FIG. 3) and the similar elasticmoduli for the two polymers indicate that these do not appear to befactors in the evolutionary retention of (VPGVG)_(n). What is now clearis that even the subtle addition of a-CH₂ -moiety, for example, whilehaving little effect on the stereochemistry of rather nonexacting,nonrestricting hydrophobic associations, has a significant effect on thethermodynamics. The larger clathrate-like cage of water surrounding theIle side chain provides a greater ΔS as the more-ordered watersurrounding the side chain becomes less ordered bulk water such that inthe transition ΔH=TΔS at a lower temperature. By means of calorimetry,the ΔH for PPP has been estimated at 5 to 6 cal/gram which isapproximately 2 kcal/mole of pentamers. Thus, the increase in entropychange need only be about 5% to cause the temperature of the transitionto decrease about 15° C. from 298° K. to 283° K. Utilizing knownhydrophobicity scales for amino acids, the hydrophobicities given in afree energy of transfer scale of kcal/mole, are -4.10 for VPGVG and-5.38 for IPGVG. While the extent of the hydrophobicity that is utilizedis expected to depend on the stereochemistry of the more-orderedpolypeptide state, it would appear that not all of the total potentialeffect is actually realized.

In accordance with another aspect of the present invention, it has alsonow been found that the abovedescribed hydrophobic effect upontransition temperatures is also supported by the elastinpolytetrapeptide, (Val¹ -Pro² -Gly³ -Gly⁴)_(n). That is, it has alsobeen discovered that high molecular weight PTP undergoes a reversibletemperature elicited aggregation with an onset of aggregation at 48° C.,rather than 24° C. as for high molecular weight PPP.

However, it has also been found that the inverse temperature transitionfor PTP is only complete at about 70° C. Moreover, this high temperatureof transition appears to be explained by considering the lowerhydrophobicity of PTP as compared to PPP.

For example, utilizing the Bull-Breese hydrophobicity scales with thehydrophobicity of the Gly residue taken as zero, the free energy oftransfer for the pentamer, VPGVG, would be -4100 cal/mole whereas thatof the tetramer, VPGG, would be -2540 cal/mole. Thus, if hydrophobicityof the repeating unit is the determining factor, then the inversetemperature transition for the PTP would be at a higher temperature thanthat of the PPP. Furthermore if the inverse temperature transition (theincrease in intramolecular order) is required for the development ofelastomeric force, then the temperature dependence of elastomeric forceof the PTP matrix would be expected to show a similar shift to highertemperature relative to that of the PPP matrix.

This inverse temperature transition is actually centered at near 50° C.for PTP, shifted some 25° C. higher than that of PPP. For Ile¹ -PTP, itis shifted some 30° C. lower in temperature than that of PTP. Also, ithas been found that the development of elastomeric force upon raisingthe temperature is similarly shifted about 25° C. higher for the PTPmatrix (20 Mrad cross-linked) as compared to the PPP matrix (20 Mradcross-linked).

Accordingly, in view of the above, it is now possible, by selecting theappropriate combination of PTP and PPP matrices or analogs thereof ofthe present invention to shift the transition temperature of abioelastomer containing elastin PTP, PPP and analogs thereof and PHPover a range of about 75° C. Furthermore, whereever this transitionwould occur in the range of about -25° C. for Phe¹ -PPP inwater/ethylene glycol or about 50° C. for PTP, in water, for example,there is a large change in elastomeric force which accompanies arelatively small change in temperature.

Thus, it is now possible to provide bioelastomers having incorporatedtherein repeating units having decreased hydrophobicity, suchas--(VPGG)_(n) --.

In particular, in accordance with the present invention, is alsoprovided a bioelastomer containing elastomeric units comprisingtetrapeptide, or pentapeptide or units thereof modified by hexapeptiderepeating units and mixtures thereof, wherein the repeating unitscomprise amino acid residues selected from the group consisting ofhydrophobic amino acid and glycine residues, wherein said repeatingunits exist in a conformation having a β-turn which comprises atetrapeptide of the formula:

    --X.sup.3 --(VPGG).sub.n --Y.sup.3 --

wherein

X³ is PGG, GG, G or a covalent bond;

Y³ is VPG, VP, V or a covalent bond; and

V is a peptide-producing residue of L-valine;

P is a peptide-producing residue of L-proline; and

G is a peptide-producing residue of glycine;

and n is an integer from 1 to 200, or n is 0, with the proviso that X³and Y³ together constitute a repeating tetrameric unit in an amountsufficient to adjust the development of elastomeric force of thebioelastomer to a predetermined temperature.

Moreover, the present invention also further provides a bioelastomercontaining elastomeric units comprising tetrapeptide, or pentapeptide orunits thereof modified by hexapeptide repeating units and mixturesthereof, wherein the repeating unit comprises amino acid residuesselected from the group consisting of hydropholic amino acid and glycineresidues, wherein said repeating units exist in a conformation having aβ-turn which comprises

(A) a polypentapeptide of the formula:

    --X.sup.1 --(IPGVG).sub.n --Y.sup.1 --

wherein X¹, Y¹, P, G, I, V and n are as defined above; and

(B) a polypentapeptide of the formula:

    --X.sup.2 --(VPGVG).sub.n --Y.sup.2 --

wherein X², Y², P, G, V and n are as defined above; or

(C) a polytetrapeptide of the formula:

    --X.sup.3 --(VPGG).sub.n --Y.sup.3 --

wherein X³, Y³, P, G, V and n are as defined above in relative amountssufficient to adjust the development of elastomeric force of saidbioelastomer to a predetermined temperature.

In accordance with the present invention are also provided PTP analogs,such as Ile¹ -PTP, which are analogous to the various PPP analogsdescribed above. In fact, any PTP analog can be used in the preparationof the present bioelastomers which suffices to attenuate thehydrophobicity of the functional repeating unit, such as --IPGG--_(n),while retaining the elasticity of the bioelastomer. Accordingly, in viewof the principles set out above, one skilled in the art would, in viewof this disclosure, be able to ascertain other PTP analogs which can beused advantageously in accordance with the present invention.

Thus, in accordance with the present invention is also provided abioelastomer containing elastomeric units comprising tetrapeptide, orpentapeptide or units thereof modified by hexapeptide repeating unitsand mixtures thereof, wherein the repeating units comprise hydrophobicamino acid and glycine residues, wherein the repeating units exist in aconformation having a β-turn which comprises a tetrapeptide of theformula:

    --X.sup.4 --(IPGG).sub.n Y.sup.4 --

wherein

X⁴ is PGG, GG, G or a covalent bond;

Y⁴ is IPG, IP, I or a covalent bond; and

I is a peptide-producing residue of L-isoleucine;

P is a peptide-producing residue of L-proline; and

G is a peptide-producing residue of glycine; and n is an integer from 1to 200, or n is 0, with the proviso that X⁴ and Y⁴ together constitute arepeating tetrameric unit, in an amount sufficient to adjust thetemperature of which the elastomeric force of the bioelastomer develops.

Of course, also within the ambit of the present invention arebioelastomers having the above-recited structural features, but whichhave any combination of the repeating units --IPGVG--_(n),--VPGVG--_(n), --VPGG--_(n), --IPGG--_(n) or other analogs thereof, suchas Ala³ -PPP or Phe¹ -PPP.

In fact, the present invention includes, in general, all bioelastomerscontaining elastomeric units comprising tetrapeptide, or pentapeptide orunits thereof modified by hexapeptide repeating units and mixturesthereof, wherein the repeating units comprise hydrophobic amino acidresidues and glycine residues, wherein the repeating units exist in aconformation having a β-turn which comprises a tetrapeptide orpentapeptide unit or repeating unit thereof, in an amount sufficient toadjust the development of elastomeric force of said bioelastomer to apredetermined temperature, with the proviso that the elasticity of thebioelastomer is retained.

However, in order to clarify the various aspects of the presentinvention relating to PTP, the following Examples and discussion areprovided. Of course, the Examples are for purposes of illustration onlyand are not intended to limit the present invention.

EXAMPLES Peptide Synthesis

General Approach: The synthesis of polytetrapeptide, (VPGG)_(n), can beachieved using any of the following permutations as the startingtetramer unit: Val-Pro-Gly-Gly, Gly-Val-Pro-Gly, Gly-Gly-Val-Pro, orPro-Gly-Gly-Val. The first sequence (VPGG) was used in this laboratoryboth with the pentachlorophenyl ester (OPcp) activation and with thep-nitrophenyl ester (ONp) activation methods, and the latter methodyielded polymer of significantly higher molecular weight. The sequence(GVPG) was utilized with --OPcp activation but no mention was made aboutthe size of the polymer. In synthesizing the polypentapeptide,(VPGVG)_(n), using different permutations of the pentamer unit withdifferent activating groups for polymerization, it was observed that thepentamer having Pro as the C-terminal amino acid and --Onp foractivation gave high molecular weight polymers. Similar results havebeen experienced in the case of the preparation of polyhexapeptide,(VAPGVG)_(n). Hence, a similar approach was determined to be reasonablein the case of PTP also, i.e., sequence (GGVP) with --ONp activation.For comparison, H--VPGG--ONp, H--GVPG--ONp and H--GGVP--ONp were alltried for polymerization. As expected, the latter tetramer sequence gavea very high molecular weight polymer when determined by the TPτ studiesand here is described the synthesis of this latter material as shown inthe Scheme II. The sequence (PGGV) was not attempted because it has anoptically active and bulky amino acid, Val, at its C-terminal. ##STR2##

Boc-GG-OBzl (I) was prepared using EDCI for coupling and washydrogenated to give the acid (II). Boc-VP-OBzl (III) was synthesized bythe mixed anhydride method in the presence of HOBt, deblocked, andcoupled with II using EDCI-HOBt to obtain Boc-GGVP-OBzl (IV). Afterhydrogenating to the acid, V, it was converted to --ONp (VI) by reactingwith bis(p-nitrophenyl)carbonate. After removing the Boc-group, theactive ester was polymerized, dialyzed against water using a 50,000molecular weight cut-off dialysis tubing and lyophilized. Theintermediate and the final products were checked by carbon-13 nuclearmagnetic resonance, thin-layer chromatography (TLC) and elementalanalyses.

Details of Syntheses: Valine and Proline are of L.configuration.Boc-amino acids were purchased from Bachem, Inc., Torrance, Calif. HOBtwas obtained from Aldrich Chemical Co., Milwaukee, Wis., and Bio-silsilica gel (200-400 mesh) was purchased from Bio-Rad Laboratories,Richmond, Calif. TLC plates were obtained from Whatman, Inc., Clifton,N.J. and the following solvent systems were used for determining thehomogeneity of the products: R_(f) ¹, CHCl₃ (C):MeOH (M):CH₃ COOH (A),95:5:3; R_(f) ², CMA (85:15:3); R_(f) ³, CMA (75:25:3); R_(f) ⁴, CM(5:1). Elemental analyses were carried out by Mic Anal, Tuscon, Ariz.Melting points were determined with a Thomas Hoover melting pointapparatus and are uncorrected.

Boc-Gly-Gly-OBzl (I): Boc-Gly-OH (17.52 g, 0.1 mole) in a mixture ofCHCl₃ (50 ml) and acetonitrile (50 ml) was cooled to -15° C. and EDCI(19.17 g, 0.1 mole) was added and stirred for 20 minutes. To this, apre-cooled solution of H-Gly-OBzl.tosylate (37.1 g, 0.11 mole), NMM(12.09 ml, 0.11 mole) in CHCl₃ (100 ml) was added and stirred overnightat room temperature. After removing the solvent, the residue was takenin CHCl₃ and extracted with acid and base. Chloroform was removed underreduced pressure, triturated with pet. ether, filtered, washed with pet.ether and dried to obtain 30.2 g of I (yield: 93.7%), m.p. 82°-83° C.R_(f) ², 0.52; R_(f) ⁴, 0.82. Anal. Calcd. for C₁₆ H₂₂ N₂ O_(5:) C,59.61; H, 6.88; N, 8.69%. Found: C, 59.43; H, 6.88; N, 8.35%.

Boc-Gly-Gly-OH (II): I (10 g, 0.31 mole) in acetic acid (100 ml) washydrogenated at 40 psi in the presence of 10% Pd-C catalyst (1 g). Thecatalyst was filtered with the aid of celite and solvent removed underreduced pressure. The residue was triturated with EtOAC, filtered,washed with EtOAC, pet. ether an dried to yield 6.3 g of II (yield:87.5%), m.p. 118°-120° C. (decomp.). R_(f) ², 0.28; R_(f) ³, 0.44. Anal.Calcd. for C₉ H₁₆ N₂ O₅.H₂ O: C, 43.19; H, 7.25; N, 11.19%. Found: C,43.53; H, 7.40; N 10.90%.

Boc-Gly-Gly-Val-Pro-OBzl (IV): III (6.0 g, 0.0148 mole) (39) wasdeblocked with HCl/Dioxane and solvent removed under reduced pressure.The residue was triturated with ether, filtered, washed with ether, thenpet. ether and dried. A very hygroscopic material was obtained (4.2 g,0.0123 mole) which was coupled in DMF with II (2.86 g, 0.0123 mole) inthe presence of 10% excess of EDCI (2.60 g) and HOBt (2.07 g). Thereaction was worked up as described for I to obtain IV as a white foamin a quantitative yield, no sharp m.p. 54°-62° C. R_(f) ², 0.42; R_(f)², 0.74. Anal. Calcd. for C₂₆ H₃₈ N₄ O₇ ; C, 60.21; H, 7.38; N, 10.80%.Found: C, 60.0; H, 7.46; N, 10.81%.

Boc-Gly-Gly-Val-Pro-OH (V): IV (6.2 g, 0.012 mole) in acetic acid washydrogenated and worked up as for II to obtain V quantitatively, nosharp m.p. 74°-83° C. R_(f) ³, 0.25; R_(f) ⁴, 0.15. Anal. Calcd. for C₁₉H₃₂ N₄ O₇ : C, 51.10; H, 7.67; N, 12.54%. Found: C, 51.28: H, 7.50N,12.38%.

Boc-Gly-Gly-Val-Pro-ONp (VI): V (5.3 g, 0.0123 mole) in pyridine (30 ml)was reacted with bis(p-nitrophenyl)carbonate (5.64 g, 0.0185 mole).After removing the solvent, the residue was taken in CHCl₃ and extractedwith acid and base. The peptide was chromatographed over a silica-gelcolumn and eluted with 35% acetane in CHCl₃ after initially eluting withCHCl₃, to obtain 4.7 g of VI (yield: 69.2%), no sharp m.p. 74°-79° C.R_(f) ², 0.76; R_(f) ⁴, 0.75. Anal. Calcd. for C₂₅ H₃₅ N₅ O₉.1/2H₂ O: C,53.75; H, 6.49; N, 12.53%. Found: C, 53.69; H, 6.44; N, 12.34%.

H-(Gly-Gly-Val-Pro)_(n) -OH (VII): VI (4.5 g, 0.0082 mole) in CHCl₃ (20ml) was treated with TFA (35 ml) for 30 minutes and solvent removedunder reduced pressure. The residue was triturated with ether, filtered,washed with ether, then with pet. ether and dried. The TFA salt (3.9 g,0.0069 mole) in DMSO (7.6 ml) and NMM (1.22 ml, 1.6 equiv) was stirredfor 14 days. After diluting with cold water, the polymer was dialyzed ina 50 kD cut-off dialysis tubing, changing water daily for 15 days, andthe retentate was lyophilyzed to yield 1.65 g of the polytetrapeptide(yield: 77%). The carbon-13 NMR spectrum of the polymer is given in FIG.5. The assignments are all indicated and there are no extraneous peaksthereby verifying the synthesis.

Temperature Profiles for Coacervation

Polypeptide coacervation in water is reversible aggregation to form anew phase with a distinct composition. Association occurs on raising thetemperature, disassociation on lowering the temperature. The process ofcoacervation was followed by monitoring the turbidity as a function oftemperature using a Cary 14 spectrophotometer set at 300 nm, a NeslabETP-3 temperature programmer with a 30° C./hour scan rate and an Omega199A thermocouple monitor. The sample cell was placed in a vibratingchamber (300 Hz) to keep the aggregates from settling and to facilitateequilibrium. The temperature profiles for coacervation are concentrationdependent. Dilution from a high concentration, after the highconcentration limit is reached (approximately 40 mg/ml for highmolecular weight elastomeric polypeptides), results in a shift of theturbidity profile to higher temperature.

Circular Dichroism Measurements

A Cary 60 spectropolarimeter equipped with a Model 6001 circulardichroism accessory with 330 Hz modulation of the left and rightcircular polarized beams was used to determine the circular dichroismpatterns of 5 mg PTP in one ml of deionized-distilled (quartz immersionheater) water. Because of the smaller size or the relative transparencyof the PTP aggregates (as with the cross-linked PTP matrix with arelatively small change in refractive index between solution and matrix)when compared to that of the PPP system, it was possible to use the 5mg/ml concentration for the CD studies without being compromised bylight scattering (particulate) distortions of the CD spectra. This isapparent from monitoring the negative band near 220 nm which becomesdamped and red-shifted as the particulate distortions becomesignificant.

Preparation of the Cross-linked PTP Matrix

The PTP was prepared for γ-irradiation crosslinking by dissolving 130milligrams of the peptide in 220 milligrams of water in a cryotube. Thematerial was shear oriented overnight at 40° C. in a previouslydescribed pestle-cryotube assembly. The sample was exposed toapproximately 8,000 Roentgen/min γ-irradiation at the Auburn UniversityNuclear Science Center. Exposure was of sufficient time to achieve a20×10⁶ radiation absorbed dose (20 Mrad).

Thermoelasticity Measurements

Thermoelasticity studies were carried out on a stres-strain apparatus.Clamping of the sample in the holder was done in two stages to preventdamage to the material at the clamp edge. The sample was first grippedlightly with the top clamp, raised to 60° C. while submerged in waterwithin the temperature jacket and allowed to equilibrate for about 2hours. The measured force consisting of the weight of the sample andgrips in water were set to zero. The bottom grip was then attached tothe sample and both grips tightened to hold the sample firmly. Thebottom clamp was driven as in a stress-strain measurement and stopped at40% elongation. Force data were recorded in 5° C. steps starting at 70°C. and continuing to 40° C. where the force approached zero.

RESULTS Temperature Profiles for Coacervation

The polytetrapeptide is soluble in water in all proportions below 40° C.On raising the temperature above 40° C. the solution becomes turbid; onstanding settling occurs to form a dense viscoelastic phase called acoacervate. The process is readily reversible; on lowering thetemperature cloudiness clears and coacervate readily redissolves. Byfollowing the turbidity as a function of temperature, temperatureprofiles for coavervation are obtained which are concentrationdependent. As more concentrated solutions are used, the onset ofturbidity occurs at lower temperatures until further increases ofconcentration cause no further lowering of the temperature for onset ofturbidity. The lower concentration above which raising the concentrationno further lowers the temperature for onset of turbidity is called thehigh concentration limit. For this high moelcular weight PTP the highconcentration limit is 40 mg/ml as 100 mg/ml gives the same profile.Dilution from 40 mg/ml causes a shift to higher temperature for theonset. These data are given in FIG. 6 where they are compared to similardata for the PPP. The midpoint for the high concentration limit of PTPis 49° C. whereas the value for the high concentration limit of PPP is25° C. The decreased hydrophobicity of the tetramer results in a 24° C.increase in the temperature required to bring about the hydrophobicinteractions attending aggregation.

Circular Dichroism

The CD spectra are shown in FIG. 7 at 40° C. (curve a) and 65° C.(curvebb) for 5 mg/ml of PTP in water. At the lower temperature there isa negative band near 220 nm and a second negative band in the 195-200 nmrange. This latter band is considered to be indicative of polypeptideswith limited order as fully disordered polypeptides are considered tohave a negative band near 195 nm with an ellipticity of -4×10⁴. Thelower magnitude of the short wavelength negative band for PTP and thenegative band near 220 nm indicate some order in the PTP at 35° C. Onraising the temperature the short wavelength negative band decreases inmagnitude indicative of a transition toward greater intramolecularorder. This transition is shown in FIG. 8A. Interestingly, its midpointcorresponds approximately to the midpoint in the temperature profile forcoacervation (see FIG. 6, curve c) for a comparable concentration. It isimportant to note for the PTP that the change in intramolecular orderprecedes the intermolecular interactions, i.e., begins at asubstantially lower temperature than the aggregational process followedin FIG. 6. For comparison in FIG. 7 are the CD spectra for PPP where ananalogous change in spectra is observed. In this case, however, thenegative band near 195 nm is much more intense making the transitiontoward greater order on raising the temperature more apparent. In FIG.8A is the inverse temperature transition of PPP plotted for comparisonwith the PTP transition. As with the aggregational data (see FIG. 6),the temperature midpoint for the PTP intramolecular transition isshifted some 25° to higher temperatures from that of the PPP. Thus, theintramolecular ordering of the PTP is shifted to higher temperature dueto the decreased hydrophobicity of the tetramer as compared to thepentamer.

Thermoelasticity Data

The temperature dependence of elastomeric force (thermoelasticity data)is plotted in FIG. 8B for 20 Mrad cross-linked PTP at an extension of40%. There is very little elastomeric force exhibited by this matrixbelow 40° C. As the temperature is raised above 40° C., however, theelastomeric force develops to a maximal value near 70° C. Also includedfor comparison in FIG. 8B are the thermoelasticity data for a 20 Mradcross-linked PPP matrix which exhibit a similar transition but shiftedsome 20° to 25° C. to lower temperatures. The development of elastomericforce, just as the temperature dependence of coacervation (see FIG. 6)and of ellipticity for the PTP, is shifted by abot 25° C. from that ofthe PPP. These properties are a function of the hydrophobicity of therepeating unit. Of particular interest is the comparison of theellipticity data for the PTP with the thermoelasticity for the PTP ofFIG. 8. The transition as followed by ellipticity, which is a measure ofintramolecular order, begins in the range 35° to 40° C., and similarlythe elastomeric force begins to develop just below 40° C. By bothphysical measurements the transition is essentially complete by 70° C.There is a close parallel between increase in intramolecular order andincrease in elastomeric force. As the aggregational intermolecularprocesses, followed by turbidity, do not become significantly untilnearly 50° C., it appears that the PTP matrix allows a delineationbetween intramolecular and intermolecular processes as related toorigins of elastomeric force.

The structural features of PTP appear to be very similar to those ofPPP. For example, it is clear that the same principles are operative asfor the PPP. The Type II Pro² -Gly³ β-turn is dominant secondarystructural feature and the ordering process is that of an inversetemperature transition with the optimization of intramolecularhydrophobic interactions as the temperature is raised. The perspectiveis again an open helix with β-turn spacers between turns of the spiraland with the Val and Pro side chains providing the intramolecularhydrophobic contacts. The suspended segment will necessarily be shorterand the librational motion will be focused on the Gly⁴ -Val¹ peptidemoiety. Based on the cyclic conformational correlate there will beapproximately 4 tetramers per turn of PTP β-spiral as opposed to theapproximately 3 pentamers per turn for the PPP β-spiral.

Effect of Repeat Unit Hydrophobicity

That the transitions toward increased elastomeric force are actuallyinverse temperature transitions dependent on the hydrophobicity of theconstituent peptide is apparent from the direction of the shift of thetransition on changing the hydrophobicity of the repeating unit. As therepeating unit becomes more hydrophobic, the temperature for thetransition shifts to lower values. Using the Nozaki-Tanford-Bull-Breesehydrophobicity scale, the pentamer (VPGVG) would have a free energy fortransfer of -4100 cal/mole whereas that for the tetramer (VPGG) would be-2540 cal/mole. For the transition ΔH=TΔS, and for a given ΔH a highertemperature would be required if the hydrophobicity giving rise to ΔSwere less. The data of FIGS. 7 and 8 show that the decreasedhydrophobicity of the tetramer requires a higher temperature for thetransition than for the more hydrophobic pentamer. This finding is inaccordance with the above-mentioned results obtained with Ile¹ -PPP.When (IPGVG)_(n), or Ile¹ -PPP, is prepared, the Ile¹ -PPP coacervates;it increases intramolecular order on increasing the temperature and theIle¹ -PPP matrix increases elastomeric force on raising the temperaturebut the transition is shifted to 9° C. The hydrophobicity for thispentamer, (IPGVG), is -5380 cal/mole. In the scale plotted as FIG. 9 isthe comparison of the temperature of the transition for the threepolypeptide elastomers and the hydrophobicities of the repeating unit.Not only is the direction of the shift correct but the magnitude of theshift is also approximately correct. It is clear that the inversetemperature transition giving rise to the intramolecular ordering andelastomeric force development is indeed proportional to thehydrophobicity of the repeating unit, and from the detailed comparisonof the transitions in FIGS. 7 and 8, it is the intramolecular processutilizing hydrophobic interactions that is responsible for thedevelopment of elastomeric force.

Thus, the bioelastomers of the present invention can encompass a widevariety of functional repeating units in order to provide a widevariation in temperature of elastomeric force development.

For example, the present bioelastomers include those having any of thefollowing repeating units as defined above:

    --X.sup.1 --(IPGVG).sub.n --Y.sup.1

    --X.sup.2 --(VPGVG).sub.n --Y.sup.2 --

    --X.sup.3 --(VPGG).sub.n --Y.sup.3 --

    --X.sup.4 --(IPGG).sub.n --Y.sup.4 --

alone or in combination with each other in order to impart to thebioelastomer a capability of developing near maximum elastomeric forceat a predetermined temperature.

However, also included with in the ambit of the present invention areall analogs of PPP and PTP and combinations thereof which modulate thehydrophobicity of the PPP and PTP repeating unit or units, withoutunduly interfering with either the formation of the viscoelastic phaseor the librational motion of the polypeptide, i.e., the elasticity.

Other examples of such analogs and combinations thereof are suchsequences as: ##STR3## where Q is either a direct covalent bond or aninterspersing amino acid residue or residues, which can be any suchresidue which does not interfere with the elasticity of the polypeptide.

Of course, the repeating pentapeptide sequence, as well as the repeatingtetrapeptide sequence can be widely substituted to modify the repeatingunit hydrophobicity, as long as the elasticity of the bioelastomer isretained. For example, the incorporated pentapeptide repeating unit canbe of the general formula:

    --R.sub.1 PR.sub.2 R.sub.3 --.sub.n

wherein R₁ is a peptide-producing residue selected from the group ofPhe, Leu, Ile and Val; R₂ is such a residue selected from the group ofAla and gly; and R₃ is selected from the group consisting of Phe, Leu,Ile and Val; and n is an integer of from 1 to about 200; and wherein Pis a L-proline-producing residue and G is a glycine-producing residue.Thus, "homopolymers" of the above pentameric sequence can be utilized or"copolymers" of the above sequence can be used in conjunction with otherrepeating units in keeping with this invention.

Also, in general, tetrapeptide repeating units of the formula:

    --R.sub.1 PGG--.sub.n

can be utilized, wherein R₁ and n are as defined above for thepentameric sequences. These units are incorporated into the presentbioelastomers in an amount sufficient to adjust the development ofelastomeric force of the bioelastomer to a predetermined temperature.

Generally, in accordance with any of the bioelastomers of the presentinvention, the bioelastomers can be a "homopolymer" of a functionalrepeating unit, such as Phe¹ -PPP, Ala³ -PPP Ile¹ -PPP, or Ile¹ -PTP; orthey can be a "copolymer" of the general formula --S_(a) --T_(b) --_(n)wherein either S or T constitutes a functional repeating unit designedto modify or shift the temperature of elastomeric force development ofthe bioelastomer, while either S or T, whichever is remaining,constitutes another repeating unit of the bioelastomer. As noted, such"copolymers" can be of either the block or random variety, which meansthat a and b can each be 1 or a larger integer.

Further, for these "copolymers", it is possible, as noted, that morethan one functional repeating unit can be used to modify the temperatureof elastomeric force development. Thus, both units --S-- and --T-- inthe formula above would be such repeating units, for example, --IPGVG--and --VPGVG--. Of course, each of S and T may be comprised of a subsetof repeating units of S_(i), S_(ii) and S_(iii). For example, three Ssubsets might be PPP analogs, such as (IPGVG), (FPGVG), where F is theone letter abbreviation for Phe, or (VPAVG).

Each one of the S or T repeating units is preferably incorporate withinthe molar range of 1-99%. More preferably still, is the incorporation ofthese units within the molar range of 5-95%. However, the actual molarcontent of any number of different repeating units is directlyproportional to the desired transition temperatures using thehydrophobicity scales as in FIG. 9.

The present bioelastomers can be used advantageously in a number ofdifferent ways which will now be described.

First, the present bioelastomers can be used in the preparation ofsynthetic vascular tissue or vascular prostheses. In general, suchsynthetic materials may be constructed in accordance with the proceduresof U.S. Pat. Nos. 4,485,227 and 4,550,447, both of which areincorporated herein in their entirety.

Additionally, it appears that the bioelastomers of the present inventioncan be utilized in the preparation of high-frequency piezoelectricdevices, utilizing the combination of a dielectric relaxation cell, afrequency source, and a force measuring device.

Essentially, a dielectric relaxation cell may be constructed inaccordance with conventional techniques, except that when using thepresent bioelastomers, a continuous rectangular slot passes through thecell. The γ-irradiation cross-linked elastomer passes through the slotand is attached at each end for force measurement.

Additionally, it has been found that the bioelastomers of the presentinvention can be utilized in the preparation of high-frequencypiezoelectric devices. In particular, at frequencies of about 10 MHz,the present bioelastomer peptides can follow an alternating electricfield, whereby saturation and synchronization of the librational motionof the elastomers, which is considered responsible for the elastomericforce thereof, would have a profound effect on the magnitude of theelastomeric force. This, in essence, is a high-frequency piezoelectriceffect. Further, the bioelastomers of the present invention have therequisite properties of elasticity and high dielectric permittivity and,on stretching, would have an axial orientation, even without floworientation prior to cross-linking, as is also required for thepiezoelectric effect.

Accordingly, the bioelastomers of the present invention may be used inthe preparation of a piezoelectric device in accordance with theprocedures of U.S. Pat. No. 4,565,943, which is incorporated herein inits entirety.

Interestingly, it has also been found that the present bioelastomersexhibit radar-absorbing properties. In accordance therewith, thebioelastomers of the present invention may be used in the preparation ofsurfaces which are radar-absorbing in accordance with U.S. Pat. No.4,034,375, which is incorporated herein in the entirety.

Finally, as noted above, the present bioelastomers can be used in thepreparation of synthetic vascular tissue. Moreover, the bioelastomers ofthe present invention now afford synthetic vascular materials havingexceptional properties.

In particular, it has been demonstrated that certain elastin repeatingsequences are chemotactic for fibroblast cells which have the capabilityof synthesizing elastin. For example, fibroblasts from ligamentum nuchaeexhibit chemotaxis toward the elastin repeat hexapeptide sequence--VGVAPG-- with a maximal response noted at a hexapeptide concentrationof about 10⁻⁹ M in solution. Similarly, the nonapeptide repeatingsequence of --AGVPGFGVG-- and its permutation --GFGVGAGVP-- are alsochemoattractants for ligamentum nuchae fibroblasts, with maximalresponses for each sequence occurring at a nonapeptide concentration ofabout 10⁻⁹ M in solution.

Thus, in view of the above, it is specifically contemplated that thepresent bioelastomers can also contain amounts of the above hexapeptideand nonapeptide repeating sequences sufficient to render the syntheticvascular tissue chemotactic for fibroblast cells. These hexapeptide andnonapeptide repeating sequences can be incorporated into the presentbioelastomers in accordance with standard peptide-synthesizing reactionswhich are well-known to peptide chemists, and as well-exemplified forthe PPP and PTP analogs hereinabove. Of course, as noted above, thesesequences can either be added as intact hexapeptide or nonapeptidesequences to the bioelastomers, or they can be generated by thestep-wise addition of a single amino acid residue at a time bysolid-phase automated synthesis.

The precise amount of chemotactic sequences incorporated into thebioelastomer depends, of course, on the desired response intensity.However, in general, in order for the synthetic vascular tissue toexhibit sufficient fibroblastic chemotaxis, it is desirable that thetotal peptide sequence of the bioelastomer comprise at least 10⁻¹¹ molar% of the chemotactic sequences. Conversely, it will usually not benecessary to exceed about 1 molar % of the chemotactic sequences basedon the total peptide content. However, as noted this amount can easilybe adjusted by experimentation to obtain the optimal amount required.

Additionally, it is noted that the term "and units thereof modified byhexapeptide units" refers to the fact that the tetrapeptide andpentapeptide repeating units of the present bioelastomers can bemodified by the addition of hexapeptide repeating units thereto for thepurpose of enhancing the mechanical strength of the bioelastomer. Ofcourse, an example of such an added hexameric unit is the sequence--APGVGV--_(n), where A, P, V and G are as defined throughout thisdisclosure, and n is an integer of about 1 to 50, and which variesdepending upon the properties desired.

Finally, it is noted that for all of the bioelastomers of the presentinvention having incorporated therein one or more functional repeatingunits of the formula:

    --X.sup.c --(repeating unit).sub.n --Y.sup.c --

where c is an integer of from 1-4 as used throughout this disclosure,and is the same value for X and Y when used, that n is an integer offrom 1 to about 200, but can also have a value of 0, when X^(c) andY^(c) together constitute, theselves, at least one repeating unit.

The present bioelastomers contain elastomeric units, in addition to theunits which are incorporated to modify the transition temperature, whichcan be a copolymer of pentapeptide "monomer" units and modifyinghexapeptide "monomer" units; tetrapeptide "monomer" units and modifyinghexapeptide "monomer" units; or pentapeptide, tetrapeptide, and"monomer" units thereof modified by hexapeptide monomer units.

Additionally, as noted above, the elastomeric units of thesebioelastomers may also contain other hexapeptide and nonapeptidesequences which exhibit fibroblastic chemotaxis, in order to render theentire bioelastomer and synthetic vascular tissue or protheses madetherefrom chemotactic for fibroblasts.

Finally, although the value of n is the above formulas is generally 1 to200, it is possible that n can be 0, with the proviso that the unitsX^(c) and Y^(c) attached to the functional repeating unit, themselvesconstitute at least one such repeating unit, in an amount sufficient toadjust the elastomeric force development of the bioelastomer to apredetermined temperature.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A bioelastomer containing repeating unitsselected from the group consisting of elastomeric tetrapeptide, orpentapeptide or units thereof modified by hexapeptide repeating unitsand mixtures thereof, wherein said repeating units comprise amino acidresidues selected from the group consisting of hydrophobic amino acidand glycine residues, wherein said repeating units exist in aconformation having a β-turn which comprises a polypentapeptide unit ofthe formula:

    --X.sup.1 --(IPGVG).sub.n --Y.sup.1 --

wherein I is a peptide-forming residue of L-isoleucine; P is apeptide-forming residue of L-proline; G is a peptide-forming residue ofglycine; V is a peptide-forming residue of L-valine; andwherein X¹ isPGVG, GVG, VG, G or a covalent bond; Y¹ is IPGV, IPG, IP, I or acovalent bond; and n is an integer from 1 to 200, or n is 0, with theproviso that X¹ and Y¹ together constitute at least one of saidpentameric unit in an amount sufficient to adjust the development ofelastomeric force of the bioelastomer to a predetermined temperature. 2.A method of decreasing the temperature of elastomeric force developmentof a bioelastomer containing repeating units selected from the groupconsisting of elastomeric tetrapeptide, or pentapeptide or units thereofmodified by hexapeptide repeating units and mixtures thereof, whereinsaid repeating units comprise amino acid residues selected from thegroup consisting of hydrophobic amino acid and glycine residues, whereinsaid repeating units exist in a conformation having a β-turn whichcomprises a polypentapeptide unit of the formula:

    --X.sup.1 --(IPGVG).sub.n --Y.sup.1 --

wherein I is a peptide-forming residue of L-isoleucine; P is apeptide-forming residue of L-proline; G is a peptide-forming residue ofglycine; V is a peptide-forming residue of L-valine; andwherein X¹ isPGVG, GVG, VG, G or a covalent bond; Y¹ is IPGV, IPG, IP, I or acovalent bond; and n is an integer from 1 to 200, or n is 0, with theproviso that X¹ and Y¹ together constitute at least one of saidpentameric unit in an amount sufficient to adjust the development ofelastomeric force of the bioelastomer to a predetermined temperature. 3.A bioelastomer containing repeating units selected from the groupconsisting of elastomeric tetrapeptide, or pentapeptide or units thereofmodified by hexapeptide repeating units and mixtures thereof, whereinsaid repeating units comprise amino acid residues selected from thegroup consisting of hydrophobic amino acid and glycine residues, whereinsaid repeating units exist in a conformation having a β-turn whichcomprises:(A) a polypentapeptide unit of the formula:

    --X.sup.1 --(IPGVG).sub.n --Y.sup.1 --

and (B) a polypentapeptide unit of the formula:

    --X.sup.2 --(VPGVG).sub.n --Y.sup.2 --

wherein I is a peptide-forming residue of L-isoleucine; P is apeptide-forming residue of L-proline; G is a peptide-forming residue ofglycine; V is a peptide-forming residue of L-valine; and wherein X¹ andX² are each PGVG, GVG, VG, G or a covalent bond; Y¹ is IPGV, IPG, IP, Ior a covalent bond; and n is an integer from 1 to 200, or n is 0, withthe proviso that X¹ and Y¹ or X² and Y² together constitute at least oneof said pentameric unit in an amount sufficient to adjust thedevelopment of elastomeric force of the bioelastomer to a predeterminedtemperature.
 4. A method of adjusting the temperature of elastomericforce development of a bioelastomer containing repeating units selectedfrom the group consisting of elastomeric tetrapeptide, or pentapeptideor units thereof modified by hexapeptide repeating units and mixturesthereof, wherein said repeating units comprise amino acid residuesselected from the group consisting of hydrophobic amino acid and glycineresidues, wherein said repeating units exist in a conformation having aβ-turn which comprises a polypentapeptide unit of the formula:

    --X.sup.1 --(IPGVG).sub.n --Y.sup.1 --

or a polypentapeptide unit of the formula:

    --X.sup.2 --(VPGVG).sub.n --Y.sup.2 --

or a mixture thereof, wherein for the above formulas, I is apeptide-forming residue of L-isoleucine; P is a peptide-forming residueof L-proline; G is a peptide-forming residue of glycine; V is apeptide-forming residue of L-valine; andwherein X¹ and X² are each PGVG,GVG, VG, G or a covalent bond; Y¹ is IPGV, IPG, IP, I or a covalentbond; and n is an integer from 1 to 200, or n is 0, with the provisothat X¹ and Y¹ or X² and Y² together constitute at least one of saidpentameric unit in an amount sufficient to adjust the development ofelastomeric force of the bioelastomer to a predetermined temperature. 5.A bioelastomer containing repeating units selected from the groupconsisting of elastomeric tetrapeptide, or pentapeptide or units thereofmodified by hexapeptide repeating units and mixtures thereof, whereinsaid repeating units comprise amino acid residues selected from thegroup consisting of hydrophobic amino acid and glycine residues, whereinsaid repeating units exist in a conformation having a β-turn whichcomprises a tetrapeptide unit of the formula:

    --X.sup.3 --(IPGVG).sub.n --Y.sup.3 --

wherein X³ is PGG, GG, G or a covalent bond; Y³ is VPG, VP, V OR acovalent bond; and V is a peptide-producing residue of L-valine; P is apeptide-producing residue of L-proline; and G is a peptide-producingresidue of glycine; and n is an integer from 1 to 200, or n is 0, withthe proviso that X³ and Y³ together constitute at least one of saidtetrameric unit, in an amount sufficient to increase the temperature atwhich the elastomeric force of the bioelastomer develops.
 6. Abioelastomer containing repeating units selected from the groupconsisting of elastomeric tetrapeptide, or pentapeptiude or unitsthereof modified by hexapeptide repeating units and mixutres thereof,wherein said repeating units comprise amino acid residues selected fromthe group consisting of hydrophobic amino acid and glycine residues,wherein said repeating units exist in a conformation having a β-turnwhich comprises incorporating into said bioelastomer a tetrapeptide unitof the formula:

    --X.sup.3 --(IPGVG).sub.n --Y.sup.3 --

wherein X³ is PGG, GG, G or a covalent bond; Y³ is VPG, VP, V OR acovalent bond; and V is a peptide-producing residue of L-valine; P is apeptide-producing residue of L-proline; and G is a peptide-producingresidue of glycine; and n is an integer from 1 to 200, or n is 0, withthe proviso that X³ and Y³ together constitute at least one of saidtetrameric unit, in an amount sufficient to adjust the development ofelastomeric force of the bioelastomer to a predetermined temperature. 7.A bioelastomer containing repeating units selected from the groupconsisting of elastomeric tetrapeptide, or pentapeptide or units thereofmodified by hexapeptide repeating units and mixtures thereof, whereinsaid repeating units comprise amino acid residues selected from thegroup consisting of hydrophobic amino acid and glycine residues, whereinsaid repeating units exist in a conformation having a β-turn whichcomprises a polypentapeptide unit of the formula:

    --X.sup.4 --(IPGG).sub.n --Y.sup.4 --

wherein X⁴ is PGG, GG G or a covalent bond; Y⁴ is IPG, IP, I or acovalent bond; and I is a peptide-forming residue of L-isoleucine; P isa peptide-forming residue of L-proline; G is a peptide-forming residueof glycine; and n is an integer from 1 to 200, or n is 0, with theproviso that X⁴ and Y⁴ together constitute at least one of saidpentameric unit in an amount sufficient to adjust the development ofelastomeric force of the bioelastomer to a predetermined temperature. 8.A method of adjusting the temperature of elastomer force development ofa bioelastomer containing repeating units selected from the groupconsisting of elastomeric tetrapeptide, or pentapeptide or units thereofmodified by hexapeptide repeating units and mixtures thereof, whereinsaid repeating units comprise amino acid residues selected from thegroup consisting of hydrophobic amino acid and glycine residues, whereinsaid repeating units exist in a conformation having a β-turn whichcomprises a polypentapeptide unit of the formula:

    --X.sup.4 --(IPGVG).sub.n --Y.sup.4 --

wherein X⁴ is PGG, GG, G or a covalent bond; Y⁴ is IPG, IP, I or acovalent bond; and I is a peptide-forming residue of L-isoleucine; P isa peptide-forming residue of L-proline; G is a peptide-forming residueof glycine; and n is an integer from 1 to 200, or n is 0, with theproviso that X⁴ and Y⁴ together constitute at least one of saidpentameric unit in an amount sufficient to adjust the development ofelastomeric force of the bioelastomer to a predetermined temperature. 9.A bioelastomer containing repeating units selected from the groupconsisting of elastomeric tetrapeptide, or pentapeptide or units thereofmodified by hexapeptide repeating units and mixtures thereof, whereinsaid repeating units comprise amino acid residues selected from thegroup consisting of hydrophobic amino acid and glycine residues, whereinsaid repeating units exist in a conformation having a β-turn whichcomprises a tetra peptide or a polypentapeptide unit or mixture thereofor repeating units thereof, capable of adjusting the temperature atwhich elastomeric force of the bioelastomer develops, in an amountsufficient to adjust the development of elastomeric force of saidbioelastomer to a predetermined temperature, with the proviso that theelasticity of the bioelastomer is retained.
 10. A bioelastomer accordingto claim 9, which further comprises a fibroblastic chemotactichexapeptide or a nonapeptide unit or repeating units an amountsufficient to impart fibroblastic chemotactic properties to saidbioelastomer.
 11. The bioelastomer according to claim 10, wherein saidchemotactic hexapeptide unit is --VGVAPG--, and said chemotacticnonapeptide sequences are --AGvPGFGVG-- and --GFGVGAGVP--.
 12. Abioelastomer containing repeating units selected from the groupconsisting of elastomeric tetrapeptide, or pentapeptide or units thereofmodified by hexapeptide repeating units and mixtures thereof, whereinsaid repeating units comprise amino acid residues selected from thegroup consisting of hydrophobic amino acid and glycine residues, whereinsaid repeating units exist in a conformation having a β-turn whichcomprises a polypentapeptide unit of the formula:

    --R.sub.1 PR.sub.2 R.sub.3 G--.sub.n

wherein R₁ is a peptide-producing residue selected from the groupconsisting of Phe, Leu, Ils, and Val; R₂ is such a residue selected fromthe group consisting of Ala and Gly; R₃ is such a residue selected fromthe group consisting of Phe, Leu, Ile, and Val; P is aL-proline-producing residue, and G is a glycine-producing residue, and nis an integer from 1 to 200, in an amount sufficient to adjust thedevelopment of elastomeric force of said bioelastomer to a predeterminedtemperature.
 13. The bioelastomer of claim 12, which further comprises afibroblastic chemotactic hexapeptide or nonapeptide unit or repeatingunits in an amount sufficient to impart fibroblastic chemotacticproperties to said bioelastomers.
 14. A method of adjusting thetemperature of elastomeric force development of a bioelastomercontaining repeating units selected from the group consisting ofelastomeric tetrapeptide, or pentapeptide or units thereof modified byhexapeptide repeating units and mixtures thereof, wherein said repeatingunits comprise amino acid residues selected from the group consisting ofhydrophobic amino acid and glycine residues, and wherein said repeatingunits exist in a conformation having a β-turn, which comprisesincorporating into said bioelastomer a tetrapeptide or pentapeptide unitor mixtures thereof or repeating units thereof, capable of adjusting thetemperature at which elastomeric force of the bioelastomer develops, inan amount sufficient to adjust the delvelopment of elastomeric force ofsaid bioelastomer, with the proviso that the elasticity of thebioelastomer is retained.
 15. A method of adjusting the temperature ofelastomeric force development of a bioelastomer containing repeatingunits selected from the group consisting of elastomeric tetrapeptide, orpentapeptide or units thereof modified by hexapeptide repeating unitsand mixtures thereof, wherein said repeating units comprise amino acidresidues selected from the group consisting of hydrophobic amino acidand glycine residues, wherein said repeating units exist in aconformation having a β-turn which comprises a polypentapeptide unit ofthe formula:

    --R.sub.1 PR.sub.2 R.sub.3 G--.sub.n

wherein R₁ is a peptide-producing residue selected from the groupconsisting of Phe, Leu, Ils, and Val; R₂ is such a residue selected fromthe group consisting of Ala and Gly; R₃ is such a residue selected fromthe group consisting of Phe, Leu, Ile, and Val; P is aL-proline-producing residue, and G is a glycine-producing residue, and nis an integer from 1 to 200, in an amount sufficient to adjust thedevelopment of elastomeric force of said bioelastomer to a predeterminedtemperature.
 16. A bioelastomer containing repeating units selected fromthe group consisting of elastomeric tetrapeptide, or pentapeptide orunits thereof modified by hexapeptide repeating units and mixturesthereof, wherein said repeating units comprise amino acid residuesselected from the group consisting of hydrophobic amino acid and glycineresidues, wherein said repeating units exist in a conformation having aβ-turn which comprises a polypentapeptide unit of the formula:

    --R.sub.1 PGG--.sub.n

wherein R₁ is a peptide-producing residue selected from the groupconsisting of Phe, Leu, Ils, and Val; P is a L-proline-producingresidue, and G is a glycine-producing residue, and n is an integer from1 to 200, in an amount sufficient to adjust the development ofelastomeric force of said bioelastomer to a predetermined temperature.17. A method of adjusting the temperature of elastomeric forcedevelopment of a bioelastomer containing repeating units selected fromthe group consisting of elastomeric tetrapeptide, or pentapeptide orunits thereof modified by hexapeptide repeating units and mixturesthereof, wherein said repeating units comprise amino acid residuesselected from the group consisting of hydrophobic amino acid and glycineresidues, wherein said repeating units exist in a conformation having aβ-turn which comprises a polypentapeptide unit of the formula:

    --R.sub.1 PGG--.sub.n

wherein R₁ is a peptide-producing residue selected from the groupconsisting of Phe, Leu, Ils, and Val; P is a L-proline-producingresidue, and G is a glycine-producing residue, and n is an integer form1 to 200, in an amount sufficient to adjust the development ofelastomeric force of said bioelastomer to a predetermined temperature.18. The bioelastomer of claim 1, which is substantially cross-linked.19. The bioelastomer of claim 3, which is substantially cross-linked.20. The bioelastomer of claim 5, which is substantially cross-linked.21. The bioelastomer of claim 7, which is substantially cross-linked.22. The bioelastomer of claim 9, which is substantially cross-linked.23. The bioelastomer of claim 12, which is substantially cross-linked.24. The bioelastomer of claim 16, which is substantially cross-linked.